Hspc-sparing treatments for rb-positive abnormal cellular proliferation

ABSTRACT

This invention is in the area of improved compounds for and methods of treating selected RB-positive cancers and other Rb-positive abnormal cellular proliferative disorders while minimizing the deleterious effects on healthy cells, for example healthy Hematopoietic Stem Cells and Progenitor Cells (HSPCs), associated with current treatment modalities. In one aspect, improved treatment of select RB-positive cancers is disclosed using specific compounds disclosed herein. In certain embodiments, the compounds described herein act as highly selective and, in certain embodiments, short, transiently-acting cyclin-dependent kinase 4/6 (CDK 4/6) inhibitors when administered to subjects.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.16/112,362, filed Aug. 24, 2018, which is a continuation of U.S.application Ser. No. 15/387,083, filed Dec. 21, 2016, which is relatedto and claims benefit of U.S. application Ser. No. 14/214,048, filedMar. 14, 2014, now U.S. Pat. No. 9,527,857, issued Dec. 27, 2016, whichis related to and claims benefit of provisional U.S. Application No.61/798,772, filed Mar. 15, 2013, provisional U.S. Application No.61/861,374, filed on Aug. 1, 2013, provisional U.S. Application61/911,354, filed on Dec. 3, 2013, and provisional U.S. Application No.61/949,786, filed on Mar. 7, 2014. The entirety of each of theseapplications is hereby incorporated by reference for all purposes.

GOVERNMENT INTEREST

The U.S. Government has rights in this invention by virtue of supportunder Grant No. 5R44AI084284 awarded by the National Institute ofAllergy and Infectious Diseases.

FIELD

This invention is in the area of improved compounds for and methods oftreating selected RB-positive cancers and other Rb-positive abnormalcellular proliferative disorders while minimizing the deleteriouseffects on healthy cells, for example healthy Hematopoietic Stem Cellsand Progenitor Cells (HSPCs), associated with current treatmentmodalities. In one aspect, improved treatment of select RB-positivecancers is disclosed using specific compounds disclosed herein. Incertain embodiments, the compounds described herein act as highlyselective and, in certain embodiments, short, transiently-actingcyclin-dependent kinase 4/6 (CDK 4/6) inhibitors when administered tosubjects.

BACKGROUND

The regulation of the cell cycle is governed and controlled by specificproteins, which are activated and deactivated mainly throughphosphorylation/dephosphorylation processes in a precisely timed manner.The key proteins that coordinate the initiation, progression, andcompletion of cell-cycle program are cyclin dependent kinases (CDKs).Cyclin-dependent kinases belong to the serine-threonine protein kinasefamily. They are heterodimeric complexes composed of a catalytic kinasesubunit and a regulatory cyclin subunit. CDK activity is controlled byassociation with their corresponding regulatory subunits (cyclins) andCDK inhibitor proteins (Cip & Kip proteins, INK4s), by theirphosphorylation state, and by ubiquitin-mediated proteolytic degradation(see D. G. Johnson, C. L. Walker, Annu. Rev. Pharmacol. Toxicol 39(1999) 295-312; D. O. Morgan, Annu. Rev. Cell Dev. Biol. 13 (1997)261-291; C. J. Sherr, Science 274 (1996) 1672-1677; T. Shimamura et al.,Bioorg. Med. Chem. Lett. 16 (2006) 3751-3754).

There are four CDKs that are significantly involved in cellularproliferation: CDK1, which predominantly regulates the transition fromG2 to M phase, and CDK2, CDK4, and CDK6, which regulate the transitionfrom G1 to S phase (Malumbres M, Barbacid M. Cell cycle, CDKs andcancer: a changing paradigm. Nat. Rev. Cancer 2009; 9(3):153-166). Inearly to mid G1 phase, when the cell is responsive to mitogenic stimuli,activation of CDK4-cyclin D and CDK6-cyclin D induces phosphorylation ofthe retinoblastoma protein (pRb). Phosphorylation of pRb releases thetranscription factor E2F, which enters the nucleus to activatetranscription of other cyclins which promote further progression of thecell cycle (see J. A. Diehl, Cancer Biol. Ther. 1 (2002) 226-231; C. J.Sherr, Cell 73 (1993) 1059-1065). CDK4 and CDK6 are closely relatedproteins with basically indistinguishable biochemical properties (see M.Malumbres, M. Barbacid, Trends Biochem. Sci. 30 (2005) 630-641).

A number of CDK 4/6 inhibitors have been identified, including specificpyrido[2,3-d]pyrimidines, 2-anilinopyrimidines, diaryl ureas,benzoyl-2,4-diaminothiazoles, indolo[6,7-a]pyrrolo[3,4-c]carbazoles, andoxindoles (see P. S. Sharma, R. Sharma, R. Tyagi, Curr. Cancer DrugTargets 8 (2008) 53-75). For example, WO 03/062236 identifies a seriesof 2-(pyridin-2-ylamino-pyrido[2,3]pyrimidin-7-ones for the treatment ofRb positive cancers that show selectivity for CDK4/6, including6-acetyl-8-cyclopentyl-5-methyl-2-(5-piperazin-1-yl-pyridin-2-ylammino)-8H-pyrido-[2,3-d]-pyrimidin-7-one(PD0332991), which is currently being tested by Pfizer in late stageclinical trials as an anti-neoplastic agent against estrogen-positive,HER2-negative breast cancer. VanderWel et al. describe aniodine-containing pyrido[2,3-d]pyrimidine-7-one (CKIA) as a potent andselective CDK4 inhibitor (see VanderWel et al., J. Med. Chem. 48 (2005)2371-2387). WO 99/15500 filed by Glaxo Group Ltd discloses proteinkinase and serine/threonine kinase inhibitors. WO 2010/020675 filed byNovartis AG describes pyrrolopyrimidine compounds as CDK inhibitors. WO2011/101409 also filed by Novartis describes pyrrolopyrimidines with CDK4/6 inhibitory activity. WO 2005/052147 filed by Novartis and WO2006/074985 filed by Janssen Pharma disclose additional CDK4 inhibitors.WO 2012/061156 filed by Tavares and assigned to G1 Therapeuticsdescribes CDK inhibitors. WO 2013/148748 filed by Francis Tavares andassigned to G1 Therapeutics describes Lactam Kinase Inhibitors.

While selective CDK4/6 inhibitors are generally designed to targetCDK4/6-replication dependent cancers, the very fact that they inhibitCDK4/6 activity may also result in deleterious effects toCDK4/6-dependent healthy cells, for example their growth inhibition.CDK4/6 activity is necessary for the production of healthy blood cellsby the bone marrow, as healthy hematopoietic stem and progenitor cells(HSPCs) require the activity of CDK4/6 for proliferation (see Roberts etal. Multiple Roles of Cyclin-Dependent Kinase 4/6 Inhibitors in CancerTherapy. JNCI 2012; 104(6):476-487). Healthy hematopoietic stem cellsgive rise to progenitor cells which in turn give rise to all thedifferentiated components of blood as shown in FIG. 1 (e.g.,lymphocytes, erythrocytes, platelets, granulocytes, monocytes). Healthyhematopoietic cells display a gradient dependency on CDK4/6 activity forproliferation during myeloid/erythroid differentiation (see Johnson etal. Mitigation of hematological radiation toxicity in mice throughpharmacological quiescence induced by CDK4/6 inhibition. J Clin. Invest.2010; 120(7): 2528-2536). Accordingly, the least differentiated cells(e.g., healthy hematopoietic stem cells (HSCs), multi-potent progenitors(MPPs), and common myeloid progenitors (CMP)) appear to be the mostdependent on CDK4/6 activity for proliferation, and therefore the mostdeleteriously affected by the use of a CDK4/6 inhibitor to treat aCDK4/6 replication dependent cancer or other proliferative disorder.

Accordingly, there is an ongoing need for improved compounds, methods,and regimes to treat patients with select Rb-positive cancers andabnormal cellular proliferative disorders while minimizing thetreatment's effect on healthy cells such as HSPCs.

SUMMARY OF THE INVENTION

Improved compounds, methods, and compositions are provided to treatselect Rb-positive abnormal cellular proliferation including anRb-positive cancer while minimizing the treatment's deleterious effectson healthy cells, such as healthy HSPCs and other CDK4/6-replicationdependent healthy cells by administration of an effective amount of acompound described herein.

In one embodiment of the invention, a compound is selected from thecompounds of Formula I, II, III, IV, or V as described herein, or apharmaceutically acceptable composition, salt, isotopic analog, orprodrug thereof. In one non-limiting example, a compound can be selectedfrom the compounds of Table 1 below, or a pharmaceutically acceptablecomposition, salt, isotopic analog, or prodrug thereof.

In one embodiment, the Rb-positive cancer can be Rb-positiveadenocarcinoma. The Rb-positive cancer can be Rb-positive adenocarcinomaof the colon. The Rb-positive cancer can also be Rb-positiveadenocarcinoma of the rectum.

Alternatively, the Rb-positive cancer can be a Rb-positive analasticastrocytoma.

The Rb-positive cancer can be Rb-positive breast cancer. In oneembodiment, the Rb-positive cancer is Rb-positive estrogen-receptorpositive, HER2-negative advanced breast cancer. Alternatively, theRb-positive cancer can be Rb-positive estrogen receptor-negative breastcancer. The Rb-positive cancer can be Rb-positive estrogen receptorpositive breast cancer. The Rb-positive cancer can be Rb-positivelate-line metastatic breast cancer. The Rb-positive cancer can beRb-positive luminal A breast cancer. The Rb-positive cancer can beRb-positive luminal B breast cancer. The Rb-positive cancer can beRb-positive Her2-negative breast cancer or Rb-positive HER2-positivebreast cancer. The Rb-positive cancer is Rb-positive male breast cancer.In one embodiment, the Rb-positive cancer is Rb-positive progesteronereceptor-negative breast cancer. The Rb-positive cancer can beRb-positive progesterone receptor-positive breast cancer. TheRb-positive cancer can be Rb-positive recurrent breast cancer. In oneembodiment, the Rb-positive cancer is Rb-positive stage IV breastcancers. In one embodiment, the Rb-positive cancer is Rb-positiveadvanced HER2-positive breast cancer.

The Rb-positive cancer can be Rb-positive bronchial cancer. TheRb-positive cancer can be Rb-positive colon cancer. The Rb-positivecancer can be Rb-positive recurrent colon cancer. The Rb-positive cancercan be Rb-positive stage IV colon cancers. In one embodiment, theRb-positive cancer is Rb-positive colorectal cancer.

In one embodiment, the Rb-positive cancer is Rb-positive endometrialcancer.

The Rb-positive cancer can be Rb-positive extragonadal seminoma. TheRb-positive cancer can be Rb-positive stage III extragonadal seminoma.The Rb-positive cancer can be Rb-positive stage IV extragonadalseminoma.

The Rb-positive cancer can be Rb-positive germ cell cancer. TheRb-positive cancer can be Rb-positive central nervous system germ celltumor. The Rb-positive cancer can be Rb-positive familial testiculargerm cell tumor. The Rb-positive cancer can be Rb-positive recurrentgonadal germ cell tumor. The Rb-positive cancer can be Rb-positiverecurrent extragonadal non-seminomatous germ cell tumor. The Rb-positivecancer can be Rb-positive extragonadal seminomatous germ cell tumor. TheRb-positive cancer can be Rb-positive recurrent malignant testiculargerm cell tumors. The Rb-positive cancer can be Rb-positive recurrentovarian germ cell tumors. The Rb-positive cancer can be Rb-positivestage III malignant testicular germ cell tumors. The Rb-positive cancercan be Rb-positive stage III ovarian germ cell tumors. The Rb-positivecancer can be Rb-positive stage IV ovarian germ cell tumors. TheRb-positive cancer can be Rb-positive stage III extragonadalnon-seminomatous germ cell tumors. The Rb-positive cancer can beRb-positive stage IV extragonadal non-seminomatous germ cell tumors. Inone embodiment, the Rb-positive cancer is Rb-positive germ cell cancer.In one embodiment, the Rb-positive cancer is Rb-positivecisplatin-refractory, unresectable germ cell cancer.

In one embodiment, the Rb-positive cancer is Rb-positive glioblastoma.

In one embodiment, the Rb-positive cancer is Rb-positive liver cancer.The Rb-positive cancer can be Rb-positive hepatocellular cancer.

The Rb-positive cancer can be Rb-positive lung cancer. In oneembodiment, the Rb-positive cancer is Rb-positive non-small cell lungcancer. In one embodiment, the Rb-positive cancer is Rb-positive KRASmutant non-small cell lung cancer.

The Rb-positive cancer can be Rb-positive melanoma. In one embodiment,the Rb-positive cancer is Rb-positive recurrent melanomas. In oneembodiment, the Rb-positive cancer is Rb-positive stage IV melanomas.

The Rb-positive cancer can be Rb-positive ovarian cancer. In oneembodiment, the Rb-positive cancer is Rb-positive ovarian epithelialcarcinoma.

The Rb-positive cancer can be Rb-positive pancreatic cancer.

The Rb-positive cancer can be Rb-positive prostate cancer.

In one embodiment, the Rb-positive cancer is Rb-positive rectal cancer.The Rb-positive cancer can be Rb-positive recurrent rectal cancer. TheRb-positive cancer can be Rb-positive stage IV rectal cancers.

The Rb-positive cancer can be Rb-positive sarcoma. The Rb-positivecancer can be Rb-positive gliosarcoma. The Rb-positive cancer can beRb-positive liposarcoma. The Rb-positive cancer can be Rb-positivefibrosarcoma. The Rb-positive cancer can be Rb-positive myxosarcoma. Inone embodiment, the Rb-positive cancer can be Rb-positivechondrosarcoma. The Rb-positive cancer can be Rb-positive osteosarcoma.

The Rb-positive cancer can be Rb-positive malignant fibroushistiocytoma. The Rb-positive cancer can be Rb-positive hemangiosarcoma.The Rb-positive cancer can be Rb-positive angiosarcoma. The Rb-positivecancer can be Rb-positive lymphangiosarcoma. The Rb-positive cancer canbe Rb-positive mesothelioma. The Rb-positive cancer can be Rb-positiveleiomyosarcoma. The Rb-positive cancer can be Rb-positiverhabdomyosarcoma. The Rb-positive cancer can be a Rb-positivemeningioma. The Rb-positive cancer can be a Rb-positive schwannoma.

In one embodiment, the Rb-positive cancer is a Rb-positivepheochromocytoma. The Rb-positive cancer can be a Rb-positive Islet cellcarcinoma. The Rb-positive cancer can be Rb-positive carcinoid. TheRb-positive cancer can be a Rb-positive paraganglioma.

In one embodiment, the Rb-positive cancer is Rb-positive squamous cellcarcinoma. The Rb-positive cancer can be Rb-positive adenocarcinoma. TheRb-positive cancer can be Rb-positive hepatocellular carcinoma. TheRb-positive cancer can be Rb-positive renal cell carcinoma. TheRb-positive cancer can be Rb-positive cholangiocarcinoma.

The Rb-positive cancer can be Rb-positive refractory solid tumors.

The Rb-positive cancer can be Rb-positive neuroblastoma.

The Rb-positive cancer can be Rb-positive medulloblastoma.

In one embodiment, the Rb-positive cancer is a Teratoma. The Rb-positivecancer can be Rb-positive ovarian immature teratoma. The Rb-positivecancer can be a Rb-positive ovarian mature teratoma. The Rb-positivecancer can be a Rb-positive ovarian specialized teratoma. TheRb-positive cancer can be Rb-positive testicular immature teratoma. TheRb-positive cancer can be Rb-positive testicular mature teratoma. TheRb-positive cancer can be Rb-positive teratoma. The Rb-positive cancercan be Rb-positive ovarian monodermal teratoma.

The Rb-positive cancer can be Rb-positive testicular cancer.

In one embodiment, the Rb-positive cancer is Rb-positive vaginal cancer.

In one embodiment, the Rb-positive cancer is selected from anRb-positive carcinoma, sarcoma, including, but not limited to, lungcancer, bone cancer, pancreatic cancer, skin cancer, cancer of the heador neck, cutaneous or intraocular melanoma, uterine cancer, ovariancancer, rectal cancer, cancer of the anal region, stomach cancer, coloncancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, cancer of the esophagus, cancer of thesmall intestine, cancer of the endocrine system, cancer of the thyroidgland, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,prostate cancer, cancer of the bladder, cancer of the kidney or ureter,renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of thecentral nervous system (CNS), primary CNS lymphoma, spinal axis tumors,brain stem glioma, pituitary adenoma, or a combination of one or more ofthe foregoing cancers.

In one embodiment, the subject is suffering from a Rb-positive abnormalcellular proliferation disorder. In one embodiment, the Rb-positiveabnormal cellular proliferation disorder is non-cancerous.

In certain embodiments, a compound described herein, when used to treata select Rb-positive cellular proliferation disorder, such as a cancer,allows for a rapid reentry of healthy cells into the normal cell-cycleand a fast reconstitution of damaged tissue and progeny cells such ashematological cells. In this aspect, the compounds described herein whenused to treat Rb-positive cancers eliminate, reduce, and/or minimize thedrug holidays and dose delays associated with the currentanti-neoplastic use of CDK4/6 inhibitors, allowing for the quickrecovery of damaged blood cells through the replication anddifferentiation of progenitor and parent cells. Specifically, theinvention includes administering to a patient having a cancer such as anRb-positive cancer an effective amount of a compound described herein,wherein the compound has a pharmacokinetic and enzymatic half-life thatprovides for a transient, reversible G1 arrest of CDK4/6-replicationdependent cells. The compound can be any of those described in thisapplication. Non-limiting examples of active compounds are described inTable 1, or a pharmaceutically acceptable composition, salt, isotopicanalog, or prodrug thereof as provided below.

In one embodiment, a compound described herein may be useful in animproved method for treating a cancer such as an Rb-positive cancer,wherein such methods have reduced or minimized effects onCDK4/6-replication dependent healthy cells due in part because they (i)utilize compounds that exhibit a pharmacokinetic and enzymatic half-lifewhich provide for a relatively short, transient and reversibleG1-arresting effect on CDK4/6-replication dependent healthy cells and(ii) allow for a rapid, reentry into the cell cycle for the healthycells following the cessation of administration or dissipation oftherapeutically effective levels in the subject. Using a compounddescribed herein allows for, for example, a reduction in the replicationdelay of the HSPCs due to CDK4/6 inhibition and/or acceleratedhematopoietic cell lineage recovery following cessation of CDK4/6inhibitory activity and/or reduced hematological deficiency because theutilized compound is short-acting, and reduces the length of off-cycleperiods or drug holidays associated with current CDK4/6 inhibitortreatment modalities which reduces or minimizes the facilitation oftumor drug resistance. In certain embodiments, the use of a compounddescribed herein allows for the continuous treatment of the subject overa longer time period without the need for an off-cycle or drug holiday.

The timely resumption of CDK4/6-replication dependent healthy cellproliferation is necessary for tissue repair, and an overly long periodof healthy cell cell-cycle arrest, for example HSPC cell-cycle arrest,is undesirable. Despite reports indicating that the selective CDK4/6inhibitor PD0332991 is an effective inhibitor of Rb-positive breastcancers, it has been discovered that such inhibitor may not be the mostideal compound for use as a chemotherapeutic due to the excessivemyelosuppressive effects of the compound. For example, PD0332991 has arelatively long-acting intra-cellular effect (see Roberts et al.Multiple Roles of Cyclin-Dependent Kinase 4/6 Inhibitors in CancerTherapy. JCNI 2012; 104(6):476-487 (FIG. 2A)), extending the transiencyof G1 arrest of healthy cells such as HSPCs leading to dose limitingmyelosuppression. Such a long acting effect delays, for example, theproliferation of HSPC cell lineages necessary to reconstitute thehematological cell lines that have been growth inhibited due to atreatment which may inhibit CDK4/6 activity, and thus, Rbphosphorylation in Rb competent cells. While desirable for itsanti-neoplastic effects, the long-acting G1 arrest provided by PD0332991requires an extended off-cycle period in order to reconstitute theerythroid, platelet, and myeloid cells (monocyte and granulocyte)adversely effected by acute HSPC G1-arrest in order to limitmyelosuppressive and allow a hematologic replication period. The use ofa compound described herein as anti-neoplastic agent for the treatmentof select Rb-positive cancers may eliminate, reduce, or minimize therequired length of off-cycle periods or drug holidays, allowing for alonger effective CDK4/6 inhibitory time period on the cancer over thecourse of an anti-neoplastic regime.

Thus in one embodiment, the invention includes administering a compounddescribed herein, including one selected from Table 1 in an effectiveamount to a host suffering from a Rb-positive cancer in a treatmentregime, wherein (either alone or in any combination thereof, each ofwhich is considered specifically and independently described): i) asubstantial portion of the CDK4/6-replication dependent healthy cells(e.g. at least 80% or greater), for example HSPCs, return to or approachpre-treatment baseline cell cycle activity (i.e., reenter thecell-cycle) in less than 24 hours, 30 hours or 36 hours from the lastadministration of a compound described herein in humans; ii) asubstantial portion of the CDK4/6-replication dependent healthy cells,for example HSPCs, reenter the cell-cycle synchronously in less than 24hours, 30 hours, or 36 hours from the last administration of a compounddescribed herein; (iii) the dissipation of the compound's inhibitoryeffect on CDK4/6-replication dependent healthy cells, for example HSPCs,occurs in less than 24 hours, 30 hours, or 36 hours from theadministration of the compound; (iv) a substantial portion of theCDK4/6-replication dependent healthy cells, for example HSPCs, return toor approach pre-treatment baseline cell cycle activity (i.e., reenterthe cell-cycle) in less than 24 hours, 30 hours, or 36 hours from thedissipation of the compound's CDK4/6 inhibitory effect; or (vi) asubstantial portion of the CDK4/6-replication dependent healthy cells,for example HSPCs, return to or approach pre-treatment baseline cellcycle activity (i.e. reenter the cell-cycle) within less than about 24hours, about 30 hours, or about 36 hours from the point in which theadministered compound's concentration level in the subject's blood dropsbelow a therapeutic effective concentration.

In a central embodiment of the invention, a compound described hereincan be administered in a concerted regimen with another agent such as anon-DNA-damaging, targeted anti-neoplastic agent or a hematopoieticgrowth factor agent for beneficial, additive, or synergistic effectagainst the abnormal cellular proliferation. It has been recentlyreported that the untimely administration of hematopoietic growthfactors can have serious side effects. For example, the use of the EPOfamily of growth factors has been associated with arterial hypertension,cerebral convulsions, hypertensive encephalopathy, thromboembolism, irondeficiency, influenza like syndromes and venous thrombosis. The G-CSFfamily of growth factors has been associated with spleen enlargement andrupture, respiratory distress syndrome, allergic reactions and sicklecell complications. By combining the administration of a compounddescribed herein and methods of the present invention with the timelyadministration of hematopoietic growth factors, for example, at the timepoint wherein the affected cells are no longer under growth arrest, itis possible for the health care practitioner to decrease the amount ofthe growth factor to minimize the unwanted adverse effects whileachieving the desired therapeutic benefit. In one embodiment, the growthfactor is administered upon cessation of the effect of the inhibitoryeffect of the compound on the CDK4/6 replication dependent healthycells, for example HSPCs. Thus, in this embodiment, the use of aselective CDK4/6 inhibitor described herein in an anti-neoplastictherapeutic regime may allow the subject to receive a reduced amount ofgrowth factor because the targeted hematopoietic cells will havereentered the cell cycle quicker than when other CDK4/6 inhibitors, forexample PD0332991. In addition, allowing rapid cell-cycle reentryfollowing G1 arrest by using a compound described herein provides forthe ability to time the administration of hematopoietic growth factorsto assist in the reconstitution of hematopoietic cell lines to maximizethe growth factor effect. As such, in one embodiment, the use of acompound or method described herein is combined with the use of ahematopoietic growth factor including, but not limited to, granulocytecolony stimulating factor (G-CSF), granulocyte-macrophage colonystimulating factor (GM-CSF), thrombopoietin, interleukin (IL)-12, steelfactor, and erythropoietin (EPO), or a derivative thereof.

In one embodiment, the CDK4/6 inhibitor is administered prior toadministration of the hematopoietic growth factor. In one embodiment,the hematopoietic growth factor administration is timed so that theCDK4/6 inhibitor's effect on HSPCs has dissipated. In one embodiment,the use of a compound described herein is combined in a therapeuticregime with at least one other chemotherapeutic agent, and can be onethat does not rely on proliferation or advancement through thecell-cycle for anti-proliferative activity. Such agent may include, butis not limited to, tamoxifen, midazolam, letrozole, bortezomib,anastrozole, goserelin, an mTOR inhibitor, a PI3 kinase inhibitors, dualmTOR-PI3K inhibitors, MEK inhibitors, RAS inhibitors, ALK inhibitors,HSP inhibitors (for example, HSP70 and HSP 90 inhibitors, or acombination thereof). Examples of mTOR inhibitors include but are notlimited to rapamycin and its analogs, everolimus (Afinitor),temsirolimus, ridaforolimus, sirolimus, and deforolimus. Examples of P13kinase inhibitors include but are not limited to Wortmannin,demethoxyviridin, perifosine, idelalisib, PX-866, IPI-145, BAY 80-6946,BEZ235, RP6503, TGR 1202 (RP5264), MLN1117 (INK1117), Pictilisib,Buparlisib, SAR245408 (XL147), SAR245409 (XL765), Palomid 529, ZSTK474,PWT33597, RP6530, CUDC-907, and AEZS-136. Examples of MEK inhibitorsinclude but are not limited to Tametinib, Selumetinib, MEK162, GDC-0973(XL518), and PD0325901. Examples of RAS inhibitors include but are notlimited to Reolysin and siG12D LODER. Examples of ALK inhibitors includebut are not limited to Crizotinib, AP26113, and LDK378. HSP inhibitorsinclude but are not limited to Geldanamycin or17-N-Allylamino-17-demethoxygeldanamycin (17AAG), and Radicicol.

In certain embodiments, a compound described herein is administered tothe subject prior to treatment with another chemotherapeutic agent,during treatment with another chemotherapeutic agent, afteradministration of another chemotherapeutic agent, or a combinationthereof. In one embodiment, a compound described herein is administeredto the subject less than about 24 hours, 20 hours, 16 hours, 12 hours, 8hours, or 4 hours or less prior to treatment with the otherchemotherapeutic agent in order to sensitize the Rb-positive cancer tothe chemotherapeutic agent. In one embodiment, the compound isadministered up to 4 hours prior to treatment with the otherchemotherapeutic agent.

In one embodiment, a compound described herein is administered in amanner that allows the drug facile access to the blood stream, forexample via intravenous injection or sublingual, intraaortal, or otherefficient blood-stream accessing route. In one embodiment, a compounddescribed herein is administered in an orally administrable formulation.In other embodiments, a compound described herein is administered viatopical, transdermal, or other desired administrative routes.

In one embodiment, a compound described herein is administered to thesubject less than about 24 hours, 20 hours, 16 hours, 12 hours, 8 hours,or 4 hours or less prior to treatment with the hematopoietic growthfactor. In one embodiment, the compound is administered up to 4 hoursprior to treatment with the hematopoietic growth factor or otherchemotherapeutic agent.

The compounds useful in the present invention show a marked selectivityfor the inhibition of CDK4 and/or CDK6 in comparison to other CDKs, forexample CDK2. For example, compounds useful in the present inventionprovide for a dose-dependent G1-arresting effect on a subject'sRb-positive cancer cells, and the methods provided for herein aresufficient to afford chemotherapeutic treatment and growth inhibition ofRB-positive cancer cells while not affecting CDK4/6-replicationindependent cells.

In one embodiment, the use of a compound described herein results in theG1-arresting effect dissipates such that the subject'sCDK4/6-replication dependent healthy cells return to theirpre-administration baseline cell-cycle activity within less than about12 hours, 14 hours, 16 hours, 18 hours, 20 hours, 24 hours, 30 hours, 36hours, or 40 hours.

In one embodiment, the G1-arresting effect dissipates such that thesubject's CDK4/6-replication dependent healthy cells return to theirpre-administration baseline cell-cycle activity within less than about24 hours, 30 hours, 36 hours, or 40 hours, or within about 48 hours ofthe cessation of administration. In one embodiment, theCDK4/6-replication dependent healthy cells are HSPCs. In one embodiment,the use of a CDK4/6 inhibitor described herein results in theG1-arresting effect dissipating so that the subject's CDK4/6-replicationdependent healthy cells return to or approach their pre-administrationbaseline cell-cycle activity within less than about 24 hours, 30 hours,36 hours, 40 hours, or within less than about 48 hours from the point inwhich the CDK4/6 inhibitor's concentration level in the subject's blooddrops below a therapeutic effective concentration. In one embodiment,the G1-arresting effect dissipates so that the subject'sCDK4/6-replication dependent healthy cells return to theirpre-administration baseline cell-cycle activity within about within lessthan about 24 hours, 30 hours, 36 hours, 40 hours, or 48 hours of thefrom the point in which the CDK4/6 inhibitor's concentration level inthe subject's blood drops below a therapeutic effective concentration.

In one embodiment, a compound described herein and useful in thedescribed methods may be synchronous in its off-effect, that is, upondissipation of the G1 arresting effect, CDK4/6-replication dependenthealthy cells exposed to a compound described herein reenter thecell-cycle in a similarly timed fashion. CDK4/6-replication dependenthealthy cells that reenter the cell-cycle do so such that the normalproportion of cells in G1 and S are reestablished quickly andefficiently, within less than about 24 hours, 30 hours, 36 hours, 40hours, or within about 48 hours of the from the point in which thecompound's concentration level in the subject's blood drops below atherapeutic effective concentration.

The rapid cell-cycle reentry associated with the compound's rapidoff-effect advantageously allow for a larger number ofCDK4/6-replication dependent healthy cells to begin replicating upondissipation of the G1 arrest compared with other CDK4/6 inhibitors suchas PD0332991. Accordingly, CDK4/6-replication dependent healthy cells,such as HSPCs, can quickly begin to replicate during an off-cycle periodor during administration periods.

The use of a compound as described herein in a therapeutic regimetargeting CDK4/6-replication dependent cancers can result in reducedanemia, reduced lymphopenia, reduced thrombocytopenia, or reducedneutropenia compared to that typically expected after, common after, orassociated with treatment with currently available antineoplasticchemotherapeutic agents. The use of the compounds as described hereinmay result in a faster recovery from bone marrow suppression associatedwith long-term use of CDK4/6 inhibitors, such as myelosuppression,anemia, lymphopenia, thrombocytopenia, or neutropenia, following thecessation of use of the CDK4/6 inhibitor. In some embodiments, the useof a compound as described herein results in reduced bone marrowsuppression associated with long-term use of CDK4/6 inhibitors, such asmyelosuppression, anemia, lymphopenia, leukopenia, thrombocytopenia, orgranulocytopenias such as neutropenia.

In some embodiments, the subject or host is a mammal, including a human.The compound can be administered to the subject by any desired route,including intravenous, sublingual, buccal, oral, intraaortal, topical,intranasal, parenteral, transdermal, systemic, intramuscular, or viainhalation.

In summary, the present invention includes the following features:

A) Optimal compounds, methods, and compositions as chemotherapeuticswhich minimize the deleterious effects on CDK4/6 replication dependenthealthy cells, for example hematopoietic stem and progenitor cells(HSPCs), in a subject undergoing treatment for a select Rb-positivecancer, comprising administering an effective amount of a compound ofFormula I, II, III, IV, or V, including a compound selected from Table 1as described herein;B) Optimal compounds, methods, and compositions as chemotherapeuticswhich minimize the deleterious effects on CDK4/6 replication dependenthealthy cells, for example hematopoietic stem and progenitor cells(HSPCs), in a subject undergoing treatment for a Rb-positive cancer,comprising administering an effective amount of a selective compounddescribed herein, wherein a substantial portion of the healthy cellsreturn to or approach pre-treatment baseline cell cycle activity (i.e.,reenter the cell-cycle) within less than about 24 hours, 30 hours, 36hours, or about 40 hours from the last administration of the CDK4/6inhibitor and wherein the CDK4/6 inhibitor has an IC₅₀ concentration forCDK4 inhibition that is more than about 1500 times less than its IC₅₀concentration for CDK2 inhibition. In certain embodiments, the CDK4/6replication dependent healthy cells are HSPCs. In certain embodiments,the CDK4/6 replication dependent healthy cells are renal epithelialcells;C) Optimal compounds, methods, and composition as chemotherapeuticswhich minimize the deleterious effect on CDK4/6 replication dependenthealthy cells in a subject undergoing treatment for a Rb-positive cancercomprising administering an effective amount of a compound describedherein, wherein a substantial portion of the CDK-replication dependenthealthy cells synchronously reenter the cell-cycle within less thanabout 24 hours, 30 hours, 36 hours, or about 40 hours following thedissipation of the compound's CDK4/6 inhibitory effect, wherein thecompound has an IC₅₀ concentration for CDK4 inhibition that is more than1500 times less than its IC₅₀ concentration for CDK2 inhibition. Incertain embodiments, the CDK4/6 replication dependent healthy cells areHSPCs. In certain embodiments, the CDK4/6 replication dependent healthycells are renal epithelial cells;D) Optimal compounds, methods, and compositions as chemotherapeuticswhich minimize the deleterious effects on CDK4/6 replication dependenthealthy cells in a subject, the method comprising administering to asubject with a Rb-positive abnormal cellular proliferative disorder aneffective amount of a selective CDK4/6 inhibitor selected from the groupconsisting of a compound described herein. In certain embodiments, thesubject's healthy cells return to or approach pre-treatment baselinecell cycle activity (i.e. reenter the cell-cycle) within less than about24 hours, about 30 hours, about 36 hours, or about 40 hours from thepoint in which the compound's concentration level in the subject's blooddrops below a therapeutic effective concentration. In certainembodiments, the CDK4/6 replication dependent healthy cells are HSPCs.In certain embodiments, the CDK4/6 replication dependent healthy cellsare renal epithelial cells.E) A compound as described herein, or a pharmaceutically acceptablecomposition, salt, isotopic analog, or prodrug thereof, for use as achemotherapeutic in the treatment of an Rb-positive abnormal cellularproliferation disorder, including Rb-positive cancers;F) A compound as described herein, or a pharmaceutically acceptablecomposition, salt, isotopic analog, and prodrug thereof, for use as achemotherapeutic regimen for the treatment of an Rb-positive abnormalcellular proliferation disorder, including Rb-positive cancers, whichminimizes the deleterious effects on CDK4/6-replication dependenthealthy cells, for example HSPCs or renal cells;G) A compound as described herein, or a pharmaceutically acceptablecomposition, salt, isotopic analog, and prodrug thereof, for use incombination with hematopoietic growth factors in a subject undergoing atherapeutic regime to treat Rb-positive abnormal cellular proliferationdisorder, including Rb-positive cancers;H) Compounds as described herein, or a pharmaceutically acceptablecomposition, salt, isotopic analog, or prodrug thereof, for use incombination with a second chemotherapeutic agent in a subject undergoinga therapeutic regime to treat a Rb-positive abnormal cellularproliferation disorder, including Rb-positive cancers;I) Use of a compound described herein, or a pharmaceutically acceptablecomposition, salt, isotopic analog, or prodrug thereof, in themanufacture of a medicament for use as a chemotherapeutic to treat asubject with a Rb-positive abnormal cellular proliferation disorder,including Rb-positive cancers;J) Use of a compound described herein, or a pharmaceutically acceptablecomposition, salts, isotopic analog, or prodrug thereof, in themanufacture of a medicament for use as chemotherapeutic to treat asubject with a Rb-positive cellular proliferation disorder, including aRb-positive cancer that, when exposed to a CDK4/6 inhibitor, is growtharrested or growth inhibited;K) Processes for the preparation of therapeutic products that contain aneffective amount of a compound described herein, for use in thetreatment of a subject having a Rb-positive abnormal cellularproliferation disorder, such as cancer, and;L) A method for manufacturing a medicament selected from the compoundsdescribed herein intended for therapeutic use as a chemotherapeutic onthe treatment of a Rb-positive abnormal cellular proliferation disorder,such as a cancer, responsive to a CDK4/6 inhibitor.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic drawing of hematopoiesis showing the hierarchicalproliferation of healthy hematopoietic stem cells (HSC) and healthyhematopoietic progenitor cells with increasing differentiation uponproliferation.

FIG. 2 is a graph of EdU incorporation vs. time after administration(hours) of PD0332991 to healthy mice HSPCs and healthy myeloidprogenitor cells. PD0332991 (150 mg/kg) was administered by oral gavageto assess the temporal effect of transient CDK4/6 inhibition on bonemarrow arrest as reported in Roberts et al. Multiple Roles ofCyclin-Dependent Kinase 4/6 Inhibitors in Cancer Therapy. JCNI 2012;104(6):476-487 (FIG. 2A). As described in Example 153, a single oraldose of PD0332991 results in a sustained reduction in HSPC EdUincorporation (circles; LKS+) and myeloid progenitor cells EdUincorporation (squares; LKS−) for greater than 36 hours.

FIG. 3A is a graph of plasma drug concentration (ng/ml) vs. time afteradministration (hours) of compound T. FIG. 3B is a graph of plasma drugconcentration (ng/ml) vs. time after administration (hours) of compoundQ. FIG. 3C is a graph of plasma drug concentration (ng/ml) vs. timeafter administration (hours) of compound GG. FIG. 3D is a graph ofplasma drug concentration (ng/ml) vs. time after administration (hours)of compound U. Compounds were dosed to mice at 30 mg/kg by oral gavage(diamonds) or 10 mg/kg by intravenous injection (squares). Blood sampleswere taken at 0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 hours post dosing andthe plasma concentrations were determined by HPLC.

FIG. 4A is a graph of the percentage of cells in the G0-G1 phase of thecell cycle vs. time after washout of the compound (hours) in humanfibroblast (Rb-positive) cells. FIG. 4B is a graph of the percentage ofcells in the S phase of the cell cycle vs. time after washout of thecompound (hours) in human fibroblast (Rb-positive) cells. FIG. 4C is agraph of the percentage of cells in the G0-G1 phase of the cell cyclevs. time after washout of the compound (hours) in human renal proximaltubule epithelial (Rb-positive) cells. FIG. 4D is a graph of thepercentage of cells in the S phase of the cell cycle vs. time afterwashout of the compound (hours) in human renal proximal tubuleepithelial (Rb-positive) cells. These cellular wash out experimentsdemonstrated that the inhibitor compounds of the present invention havea short, transient G1-arresting effect in different cell types. Theeffect on the cell cycle following washing out of the compounds wasdetermined at 24, 36, 40, and 48 hours. As described in Example 155, theresults show that cells treated with PD0332991 (circles) tooksignificantly longer to reach baseline levels of cell division (seecells treated only with DMSO (diamonds)), than cells treated withcompound T (squares), compound Q (triangles), compound GG (X), orcompound U (X with cross).

FIG. 5A is a graph of the ratio of EdU incorporation into HSPCs(compared to untreated control mice) following oral gavage of compoundsT, Q, or GG at 150 mg/kg at either 12 or 24 hours post administration.FIG. 5B is a graph of the percentage of EdU positive HSPC cells for micetreated with compound T at either 12 or 24 hours. Mice were dosed with50 mg/kg (triangles), 100 mg/kg (squares), or 150 (upside downtriangles) mg/kg by oral gavage. FIG. 5C is a graph of the percentage ofEdU positive HSPC cells for mice treated with compound T (150 mg/kg byoral gavage) at either 12, 24, 36 and 48 hours. As described in Example156, compound T and GG demonstrated a reduction in EdU incorporation at12 hours, and started to return to normal levels of cell division by 24hours.

FIG. 6 is a graph of the percentage of EdU positive HSPC cells for micetreated with either PD0332991 (triangles) or compound T (upside downtriangles) v. time after administration (hours) of the compound. Bothcompounds were administered at 150 mg/kg by oral gavage and thepercentage of EdU positive HSPC cells was measured at 12, 24, 36 or 48hours. As described in Example 157, a single oral dose of PD0332991results in a sustained reduction of HSPC proliferation for greater than36 hours. In contrast, a single oral dose of Compound T results in aninitial reduction of HSPC proliferation at 12 hours, but proliferationof HSPCs resumes by 24 hours after dosage of Compound T.

FIG. 7 provides the half-life (minutes) of compound T and PD0332991 inhuman and animal (monkey, dog, rat, and mouse) liver microsomes. Asdescribed in Example 158, PD0332991 has a half-life greater than 60minutes in each of the species tested. Compound T was determined to havea shorter half-life than PD0332991 in each of the species tested.

FIG. 8 is a graph showing the tumor volume (mm³) in MMTV-c-neu(Rb-positive) tumor bearing mice treated with Compound T at 100mg/kg/day (squares) or 150 mg/kg/day (triangles) v. time afteradministration (days) of Compound T. Tumor-bearing MMTV-c-neu mice(control, n=9; compound T, 100 mg/kg, n=7; compound T, 150 mg/kg, n=6)were treated with either compound T delivered in chow or standard chow(circles). Day 0 represents the first day of compound treatment. Micewere treated with compound T for 28 days (as represented by the boxaround the numbers on the x-axis indicating days of treatmentadministration). After 28 days, all mice were fed standard chow. Tumorvolumes were recorded weekly (up to 56 days) and graphed asmean±standard error of the mean. As described in Example 159, continuoustreatment with Compound T (100 mg/kg/d or 150 mg/kg/d) led to a markedreduction in tumor volume during a 28 day course of therapy compared tocontrol.

FIG. 9 is a waterfall plot of the percentage change in MMTV-c-neu mice(Rb-positive) tumor volumes for each mouse treated with Compound T ateither 100 mg/kg (horizontal lined box) or 150 mg/kg (slanted linedbox). Tumor volumes were compared to the average tumor size of untreatedanimals on day 21. Tumor volumes for mice treated with compound Trepresent the best response seen on day 28 or beyond. Negative valuesindicate tumor shrinkage.

FIG. 10 is a table showing the objective response rate (ORR) ofMMTV-c-neu (Rb-positive) tumors in mice treated with compounds T, GG, orU in the MMTV-c-neu luminal breast cancer (Rb-positive) model. All threecompounds were administered orally via medicated diets (100 mg/kg/day).Medicated diets were administered for 28 consecutive days and thenstopped. RECIST criteria were used to assess objective response rates.The objective response rates (ORR) were categorized based on thepercentage change in tumor volume, using the following categories: CR(complete response)=100% response; PR (partial response)=at least a 30%decrease; SD (stable disease)=no change (not a PR and not a PD); and PD(progressive disease)=20% increase. As described in Example 160,continuous treatment with compounds T, GG, or U led to a markedreduction in tumor volume during a 28 day course of therapy.

FIG. 11 is a graph showing the tumor volume (mm³) in MMTV-c-neu(Rb-positive) tumor bearing mice treated with Compound T (open circles),Compound GG (diamonds), or Compound U (squares) v. time afteradministration of each compound (days). Tumor-bearing MMTV-c-neu mice(control, n=9; compound T, 100 mg/kg, n=7; compound GG, 100 mg/kg, n=7;compound U, 100 mg/kg, n=8) were treated with compound delivered in chowor standard chow (closed circles). Day 0 represents the first day ofcompound treatment. Mice were treated with compound for 28 days (asrepresented by the box around the numbers on the x-axis indicating daysof treatment administration). After 28 days, all mice were fed standardchow. Tumor volumes were recorded weekly (up to 56 days) and graphed asmean±standard error of the mean. As described in Example 160, continuoustreatment with compounds T, GG, or U led to a marked reduction in tumorvolume during a 28 day course of therapy, with compounds T and U showinga 100% objective response rate, while compound GG showed an 85%objective response rate.

FIG. 12 is a waterfall plot of the percentage change in MMTV-c-neu(Rb-positive) tumor volumes for each mouse treated with 100 mg/kgcompound T (bars with diagonal lines, n=7), 100 mg/kg compound GG (blackand white boxed bars, n=7), 100 mg/kg compound U (filled bars, n=8), orno treatment (open bars, n=9). Tumor volumes were compared to theaverage tumor size of untreated animals on day 21. Tumor volumes formice treated with compound T, GG, or U represent the best response seenon day 14 or beyond. Negative values indicate tumor shrinkage. Negativevalues indicate tumor shrinkage. As described in Example 160, continuoustreatment with compounds T, GG, or U led to a marked reduction in tumorvolume during a 28 day course of therapy.

FIGS. 13-15 illustrate several exemplary embodiments of R² of thecompounds of the invention.

FIGS. 16A-16C, 17A-D, 18A-18C, 19A-19B, and 20A-20F illustrate severalexemplary embodiments of the core structure of the compounds of theinvention.

FIG. 21 is a graph of the cellular proliferation of MCF7 (Rb-positive)cells (breast adenocarcinoma) (as measured by Relative Light Units(RLU)) v. variable molar concentration (M) of treatment with eitherPD0332991 (circles) or Compound T (Table 1; squares). The MCF7 cellswere seeded in Costar (Tewksbury, Mass.) 3903 96 well tissue culturetreated white walled/clear bottom plates. A nine point dose responsedilution series from 10 uM to 1 nM was performed and cell viability wasdetermined after six days of compound treatment as indicated using theCellTiter-Glo® luminescent cell viability assay (CTG; Promega, Madison,Wis., United States of America) following the manufacturer'srecommendations. Plates were read on a BioTek (Winooski, Vt.) Syngergy2multi-mode plate reader. The Relative Light Units (RLU) were plotted asa result of variable molar concentration and data was analyzed usingGraphpad (LaJolla, Calif.) Prism 5 statistical software to determine theEC₅₀ for each compound.

FIG. 22 is a graph of the cellular proliferation of MCF7 (Rb-positive)cells (breast adenocarcinoma) (as measured by Relative Light Units(RLU)) v. variable molar concentration (M) of treatment with eitherCompound Q (Table 1; circles) or Compound GG (Table 1; squares).Cellular proliferation was determined using the CellTiter-Glo®luminescent cell viability assay, as described in FIG. 21 and Example152.

FIG. 23 is a graph of the cellular proliferation of MCF7 (Rb-positive)cells (breast adenocarcinoma) (as measured by Relative Light Units(RLU)) v. variable molar concentration (M) of treatment with eitherCompound U (Table 1; circles) or Compound H (Table 1; squares). Cellularproliferation was determined using the CellTiter-Glo® luminescent cellviability assay, as described in FIG. 21 and Example 152.

FIG. 24 is a graph of the cellular proliferation of MCF7 (Rb-positive)cells (breast adenocarcinoma) (as measured by Relative Light Units(RLU)) v. variable molar concentration (M) of treatment with eitherCompound MM (Table 1; circles) or Compound 00 (Table 1; squares).Cellular proliferation was determined using the CellTiter-Glo®luminescent cell viability assay, as described in FIG. 21 and Example152.

FIG. 25 is a graph of the cellular proliferation of ZR75-1 (Rb-positive)cells (breast adenocarcinoma) (as measured by Relative Light Units(RLU)) v. variable molar concentration (M) of treatment with eitherPD0332991 (circles) or Compound T (Table 1; squares). Cellularproliferation was determined using the CellTiter-Glo® luminescent cellviability assay, as described in FIG. 21 and Example 152.

FIG. 26 is a graph of the cellular proliferation of ZR75-1 (Rb-positive)cells (breast adenocarcinoma) (as measured by Relative Light Units(RLU)) v. variable molar concentration (M) of treatment with eitherCompound Q (Table 1; circles) or Compound GG (Table 1; squares).Cellular proliferation was determined using the CellTiter-Glo®luminescent cell viability assay, as described in FIG. 21 and Example152.

FIG. 27 is a graph of the cellular proliferation of ZR75-1 (Rb-positive)cells (breast adenocarcinoma) (as measured by Relative Light Units(RLU)) v. variable molar concentration (M) of treatment with eitherCompound U (Table 1; circles) or Compound H (Table 1; squares). Cellularproliferation was determined using the CellTiter-Glo® luminescent cellviability assay, as described in FIG. 21 and Example 152.

FIG. 28 is a graph of the cellular proliferation of ZR75-1 (Rb-positive)cells (breast adenocarcinoma) (as measured by Relative Light Units(RLU)) v. variable molar concentration (M) of treatment with eitherCompound MM (Table 1; circles) or Compound 00 (Table 1; squares).Cellular proliferation was determined using the CellTiter-Glo®luminescent cell viability assay, as described in FIG. 21 and Example152.

FIG. 29A is a graph of the percentage of cells in G2-M phase (opencircles), S phase (triangles), G0-G1 phase (squares), <2N (diamonds) vs.variable concentration (nM) of compound T in tHS68 cells. TheCDK4/6-dependent cell line (tHS68) was treated with the indicatedconcentrations of Compound T for 24 hours. Following treatment ofCompound T, cells were harvested and analyzed for cell cycledistribution. As described in Example 161, tHS68 cells show a clean G1arrest accompanied by a corresponding decrease in the number of cells inS-phase.

FIG. 29B is a graph of the number of tHS68 cells (CDK4/6-dependent cellline) vs. the DNA content of the cells (as measured by propidiumiodide). Cells were treated with DMSO for 24 hours, harvested, andanalyzed for cell cycle distribution.

FIG. 29C is a graph of the number of WM2664 cells (CDK4/6-dependent cellline) vs. the DNA content of the cells (as measured by propidiumiodide). Cells were treated with DMSO for 24 hours, harvested, andanalyzed for cell cycle distribution.

FIG. 29D is a graph of the number of A2058 cells (CDK4/6-independentcell line) vs. the DNA content of the cells (as measured by propidiumiodide). Cells were treated with DMSO for 24 hours, harvested, andanalyzed for cell cycle distribution.

FIG. 29E is a graph of the number of tHS68 cells (CDK4/6-dependent cellline) vs. the DNA content of the cells (as measured by propidium iodide)after treatment with Compound T. Cells were treated with Compound T (300nM) for 24 hours, harvested, and analyzed for cell cycle distribution.As described in Example 161, treatment of tHS68 cells with Compound Tcauses a loss of the S-phase peak (indicated by arrow).

FIG. 29F is a graph of the number of WM2664 cells (CDK4/6-dependent cellline) vs. the DNA content of the cells (as measured by propidium iodide)after treatment with Compound T. Cells were treated with Compound T (300nM) for 24 hours, harvested, and analyzed for cell cycle distribution.As described in Example 161, treatment of WM2664 cells with Compound Tcauses a loss of the S-phase peak (indicated by arrow).

FIG. 29G is a graph of the number of A2058 cells (CDK4/6-independentcell line) vs. the DNA content of the cells (as measured by propidiumiodide) after treatment with Compound T. Cells were treated withCompound T (300 nM) for 24 hours, harvested, and analyzed for cell cycledistribution. As described in Example 161, treatment of A2058 cells withCompound T does not cause a loss of the S-phase peak (indicated byarrow).

FIG. 30 is a Western blot showing the phosphorylation levels of Rb atSer807/811 and Ser780 after treatment with Compound T. CDK4/6-dependent(tHS68 or WM2664) and CDK4/6-independent cell lines (A2058) were treatedwith Compound T (300 nM) for the indicated times (0, 4, 8, 16, and 24hours). MAPK levels are shown as a control for protein levels. Followingtreatment, cells were harvested and analyzed for Rb-phosphorylation bywestern blot analysis. As described in Example 162, Compound T treatmentresulted in reduced Rb-phosphorylation starting 16 hours after treatmentin CDK4/6-dependent cell lines (tHS68 and WM2664), but not in theCDK4/6-independent cell line (A2058).

DETAILED DESCRIPTION OF THE INVENTION

Improved compounds, methods, and compositions are provided aschemotherapeutics for the treatment of select Rb-positive cancers whichminimize or reduce the deleterious effects on CDK4/6 replicationdependent healthy cells, such as hematopoietic stem cells and/orprogenitor cells (HSPCs), due to CDK4/6 growth arrest, in subjects,typically humans.

Definitions

Unless otherwise stated, the following terms used in this application,including the specification and claims, have the definitions givenbelow. As used in the specification and the appended claims, thesingular forms “a,” “an” and “the” include plural referents unless thecontext clearly dictates otherwise. Definition of standard chemistryterms may be found in reference works, including Carey and Sundberg(2007) Advanced Organic Chemistry 5^(th) Ed. Vols. A and B, SpringerScience+Business Media LLC, New York. The practice of the presentinvention will employ, unless otherwise indicated, conventional methodsof synthetic organic chemistry, mass spectroscopy, preparative andanalytical methods of chromatography, protein chemistry, biochemistry,recombinant DNA techniques and pharmacology. Conventional methods oforganic chemistry include those included in March's Advanced OrganicChemistry: Reactions, Mechanisms, and Structure, 6^(th) Edition, M. B.Smith and J. March, John Wiley & Sons, Inc., Hoboken, N.J., 2007.

The term “alkyl,” either alone or within other terms such as “haloalkyl”and “alkylamino,” embraces linear or branched radicals having one toabout twelve carbon atoms. “Lower alkyl” radicals have one to about sixcarbon atoms. Examples of such radicals include methyl, ethyl, n-propyl,isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isoamyl,hexyl and the like. The term “alkylene” embraces bridging divalentlinear and branched alkyl radicals. Examples include methylene,ethylene, propylene, isopropylene and the like.

The term “alkenyl” embraces linear or branched radicals having at leastone carbon-carbon double bond of two to about twelve carbon atoms.“Lower alkenyl” radicals having two to about six carbon atoms. Examplesof alkenyl radicals include ethenyl, propenyl, allyl, propenyl, butenyland 4-methylbutenyl. The terms “alkenyl” and “lower alkenyl,” embraceradicals having “cis” and “trans” orientations, or alternatively, “E”and “Z” orientations.

The term “alkynyl” denotes linear or branched radicals having at leastone carbon-carbon triple bond and having two to about twelve carbonatoms. “Lower alkynyl” radicals having two to about six carbon atoms.Examples of such radicals include propargyl, butynyl, and the like.

Alkyl, alkenyl, and alkynyl radicals may be optionally substituted withone or more functional groups such as halo, hydroxy, nitro, amino,cyano, haloalkyl, aryl, heteroaryl, heterocyclo and the like.

The term “alkylamino” embraces “N-alkylamino” and “N,N-dialkylamino”where amino groups are independently substituted with one alkyl radicaland with two alkyl radicals, respectively. “Lower alkylamino” radicalshave one or two alkyl radicals of one to six carbon atoms attached to anitrogen atom. Suitable alkylamino radicals may be mono or dialkylaminosuch as N-methylamino, N-ethylamino, N,N-dimethylamino, N,N-diethylaminoand the like.

The term “halo” means halogens such as fluorine, chlorine, bromine oriodine atoms.

The term “haloalkyl” embraces radicals wherein any one or more of thealkyl carbon atoms is substituted with one or more halo as definedabove. Examples include monohaloalkyl, dihaloalkyl and polyhaloalkylradicals including perhaloalkyl. A monohaloalkyl radical, for oneexample, may have an iodo, bromo, chloro or fluoro atom within theradical. Dihalo and polyhaloalkyl radicals may have two or more of thesame halo atoms or a combination of different halo radicals. “Lowerhaloalkyl” embraces radicals having 1-6 carbon atoms. Examples ofhaloalkyl radicals include fluoromethyl, difluoromethyl,trifluoromethyl, chloromethyl, dichloromethyl, trichloromethyl,pentafluoroethyl, heptafluoropropyl, difluorochloromethyl,dichlorofluoromethyl, difluoroethyl, difluoropropyl, dichloroethyl anddichloropropyl. “Perfluoroalkyl” means an alkyl radical having allhydrogen atoms replaced with fluoro atoms. Examples includetrifluoromethyl and pentafluoroethyl.

The term “aryl”, alone or in combination, means a carbocyclic aromaticsystem containing one or two rings wherein such rings may be attachedtogether in a fused manner. The term “aryl” embraces aromatic radicalssuch as phenyl, naphthyl, indenyl, tetrahydronaphthyl, and indanyl. Morepreferred aryl is phenyl. Said “aryl” group may have 1 or moresubstituents such as lower alkyl, hydroxyl, halo, haloalkyl, nitro,cyano, alkoxy, lower alkylamino, and the like. An aryl group may beoptionally substituted with one or more functional groups such as halo,hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocycloand the like.

The term “heterocyclyl” (or “heterocyclo”) embraces saturated, andpartially saturated heteroatom-containing ring radicals, where theheteroatoms may be selected from nitrogen, sulfur and oxygen.Heterocyclic rings comprise monocyclic 6-8 membered rings, as well as5-16 membered bicyclic ring systems (which can include bridged fused andspiro-fused bicyclic ring systems). It does not include rings containing—O—O—. —O—S— or —S—S— portions. Said “heterocyclyl” group may have 1 to3 substituents such as hydroxyl, Boc, halo, haloalkyl, cyano, loweralkyl, lower aralkyl, oxo, lower alkoxy, amino, lower alkylamino, andthe like.

Examples of saturated heterocyclo groups include saturated 3- to6-membered heteromonocyclic groups containing 1 to 4 nitrogen atoms[e.g. pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl,piperazinyl]; saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.morpholinyl]; saturated 3 to 6-membered heteromonocyclic groupcontaining 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g.,thiazolidinyl]. Examples of partially saturated heterocyclyl radicalsinclude dihydrothienyl, dihydropyranyl, dihydrofuryl, dihydrothiazolyl,and the like.

Particular examples of partially saturated and saturated heterocyclogroups include pyrrolidinyl, imidazolidinyl, piperidinyl, pyrrolinyl,pyrazolidinyl, piperazinyl, morpholinyl, tetrahydropyranyl,thiazolidinyl, dihydrothienyl, 2,3-dihydro-benzo[1,4]dioxanyl,indolinyl, isoindolinyl, dihydrobenzothienyl, dihydrobenzofuryl,isochromanyl, chromanyl, 1,2-dihydroquinolyl,1,2,3,4-tetrahydro-isoquinolyl, 1,2,3,4-tetrahydro-quinolyl,2,3,4,4a,9,9a-hexahydro-1H-3-aza-fluorenyl,5,6,7-trihydro-1,2,4-triazolo[3,4-a]isoquinolyl,3,4-dihydro-2H-benzo[1,4]oxazinyl, benzo[1,4]dioxanyl,2,3-dihydro-1H-1λ′-benzo[d]isothiazol-6-yl, dihydropyranyl, dihydrofuryland dihydrothiazolyl, and the like.

Heterocyclo groups also includes radicals where heterocyclic radicalsare fused/condensed with aryl radicals: unsaturated condensedheterocyclic group containing 1 to 5 nitrogen atoms, for example,indolyl, isoindolyl, indolizinyl, benzimidazolyl, quinolyl, isoquinolyl,indazolyl, benzotriazolyl, tetrazolopyridazinyl [e.g.,tetrazolo[1,5-b]pyridazinyl]; unsaturated condensed heterocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms [e.g.benzoxazolyl, benzoxadiazolyl]; unsaturated condensed heterocyclic groupcontaining 1 to 2 sulfur atoms and 1 to 3 nitrogen atoms [e.g.,benzothiazolyl, benzothiadiazolyl]; and saturated, partially unsaturatedand unsaturated condensed heterocyclic group containing 1 to 2 oxygen orsulfur atoms [e.g. benzofuryl, benzothienyl,2,3-dihydro-benzo[1,4]dioxinyl and dihydrobenzofuryl].

The term “heteroaryl” denotes aryl ring systems that contain one or moreheteroatoms selected from the group O, N and S, wherein the ringnitrogen and sulfur atom(s) are optionally oxidized, and nitrogenatom(s) are optionally quarternized. Examples include unsaturated 5 to 6membered heteromonocyclyl group containing 1 to 4 nitrogen atoms, forexample, pyrrolyl, imidazolyl, pyrazolyl, 2-pyridyl, 3-pyridyl,4-pyridyl, pyrimidyl, pyrazinyl, pyridazinyl, triazolyl [e.g.,4H-1,2,4-triazolyl, IH-1,2,3-triazolyl, 2H-1,2,3-triazolyl]; unsaturated5- to 6-membered heteromonocyclic group containing an oxygen atom, forexample, pyranyl, 2-furyl, 3-furyl, etc.; unsaturated 5 to 6-memberedheteromonocyclic group containing a sulfur atom, for example, 2-thienyl,3-thienyl, etc.; unsaturated 5- to 6-membered heteromonocyclic groupcontaining 1 to 2 oxygen atoms and 1 to 3 nitrogen atoms, for example,oxazolyl, isoxazolyl, oxadiazolyl [e.g., 1,2,4-oxadiazolyl,1,3,4-oxadiazolyl, 1,2,5-oxadiazolyl]; unsaturated 5 to 6-memberedheteromonocyclic group containing 1 to 2 sulfur atoms and 1 to 3nitrogen atoms, for example, thiazolyl, thiadiazolyl [e.g.,1,2,4-thiadiazolyl, 1,3,4-thiadiazolyl, 1,2,5-thiadiazolyl].

The term “heteroarylalkyl” denotes alkyl radicals substituted with aheteroaryl group. Examples include pyridylmethyl and thienylethyl.

The term “sulfonyl”, whether used alone or linked to other terms such asalkylsulfonyl, denotes respectively divalent radicals —SO₂—.

The terms “carboxy” or “carboxyl”, whether used alone or with otherterms, such as “carboxyalkyl”, denotes —C(O)—OH.

The term “carbonyl”, whether used alone or with other terms, such as“aminocarbonyl”, denotes —C(O)—.

The term “aminocarbonyl” denotes an amide group of the Formula—C(O)—NH₂.

The terms “heterocycloalkyl” embrace heterocyclic-substituted alkylradicals. Examples include piperidylmethyl and morpholinylethyl.

The term “arylalkyl” embraces aryl-substituted alkyl radicals. Examplesinclude benzyl, diphenylmethyl and phenylethyl. The aryl in said aralkylmay be additionally substituted with halo, alkyl, alkoxy, halkoalkyl andhaloalkoxy.

The term “cycloalkyl” includes saturated carbocyclic groups of 3 to 10carbons. Lower cycloalkyl groups include C₃-C₆ rings. Examples includecyclopentyl, cyclopropyl, and cyclohexyl. Cycloalkyl groups may beoptionally substituted with one or more functional groups such as halo,hydroxy, nitro, amino, cyano, haloalkyl, aryl, heteroaryl, heterocycloand the like.

The term “cycloalkylalkyl” embraces cycloalkyl-substituted alkylradicals. “Lower cycloalkylalkyl” radicals are cycloalkyl radicalsattached to alkyl radicals having one to six carbon atoms. Examples ofinclude cyclohexylmethyl. The cycloalkyl in said radicals may beadditionally substituted with halo, alkyl, alkoxy and hydroxy.

The term “cycloalkenyl” includes carbocyclic groups having one or morecarbon-carbon double bonds including “cycloalkyldienyl” compounds.Examples include cyclopentenyl, cyclopentadienyl, cyclohexenyl andcycloheptadienyl.

The term “comprising” is meant to be open ended, including the indicatedcomponent but not excluding other elements.

The term “oxo” as used herein contemplates an oxygen atom attached witha double bond.

The term “nitro” as used herein contemplates —NO₂.

The term “cyano” as used herein contemplates —CN.

As used herein, the term “prodrug” means a compound which whenadministered to a host in vivo is converted into the parent drug. Asused herein, the term “parent drug” means any of the presently describedchemical compounds that are useful to treat any of the disordersdescribed herein, or to control or improve the underlying cause orsymptoms associated with any physiological or pathological disorderdescribed herein in a host, typically a human. Prodrugs can be used toachieve any desired effect, including to enhance properties of theparent drug or to improve the pharmaceutic or pharmacokinetic propertiesof the parent. Prodrug strategies exist which provide choices inmodulating the conditions for in vivo generation of the parent drug, allof which are deemed included herein. Nonlimiting examples of prodrugstrategies include covalent attachment of removable groups, or removableportions of groups, for example, but not limited to acylation,phosphorylation, phosphonylation, phosphoramidate derivatives,amidation, reduction, oxidation, esterification, alkylation, othercarboxy derivatives, sulfoxy or sulfone derivatives, carbonylation oranhydride, among others.

Throughout the specification and claims, a given chemical formula orname shall encompass all optical and stereoisomers, as well as racemicmixtures where such isomers and mixtures exist, unless otherwise noted.

The current invention is directed to an HSPC-sparing strategy during thetreatment of Rb-positive proliferation disorders. According, as usedherein, the term “HSPCs” is meant to describe healthy hematopoietic stemand/or hematopoietic progenitor cells, as opposed to diseased HSPCs orcells of related hematological origin. HSPCs include hematopoietic stemcells, such as long term hematopoietic stem cells (LT-HSCs) and shortterm hematopoietic stem cells (ST-HSCs), and hematopoietic progenitorcells, including multipotent progenitors (MPPs), common myeloidprogenitors (CMPs), common lymphoid progenitors (CLPs),granulocyte-monocyte progenitors (GMPs) and megakaryocyte-erythroidprogenitors (MEPs).

In some embodiments, a CDK4/6-replication dependent healthy cell is ahematopoietic stem progenitor cell. In some embodiments, theCDK4/6-replication dependent healthy cell may be a cell in anon-hematopoietic tissue, such as, but not limited to, the liver,kidney, pancreas, brain, lung, adrenals, intestine, gut, stomach, skin,auditory system, bone, bladder, ovaries, uterus, testicles, gallbladder,thyroid, heart, pancreatic islets, blood vessels, and the like.

The term “selective CDK4/6 inhibitor” used in the context of thecompounds described herein includes compounds that inhibit CDK4activity, CDK6 activity, or both CDK4 and CDK6 activity at an IC₅₀ molarconcentration at least about 500, or 1000, or 1500, or 1800, or 2000times less than the IC₅₀ molar concentration necessary to inhibit to thesame degree of CDK2 activity in a standard phosphorylation assay.

As used herein the term “chemotherapy” or “chemotherapeutic agent”refers to treatment with a cytostatic or cytotoxic agent (i.e., acompound) to reduce or eliminate the growth or proliferation ofundesirable cells, for example cancer cells. Thus, as used herein,“chemotherapy” or “chemotherapeutic agent” refers to a cytotoxic orcytostatic agent used to treat a proliferative disorder, for examplecancer.

By “induces G1-arrest” is meant that the inhibitor compound induces aquiescent state in a substantial portion of a cell population at the G1phase of the cell cycle.

By “hematological deficiency” is meant reduced hematological celllineage counts or the insufficient production of blood cells (i.e.,myelodysplasia) and/or lymphocytes (i.e., lymphopenia, the reduction inthe number of circulating lymphocytes, such as B- and T-cells).Hematological deficiency can be observed, for example, asmyelosuppression in form of anemia, reduction in platelet count (i.e.,thrombocytopenia), reduction in white blood cell count (i.e.,leukopenia), or the reduction in granulocytes (e.g., neutropenia).

By “synchronous reentry into the cell cycle” is meant thatCDK4/6-replication dependent healthy cells, for example HSPCs, inG1-arrest due to the effect of a CDK4/6 inhibitor compound reenter thecell-cycle within relatively the same collective timeframe or atrelatively the same rate upon dissipation of the compound's effect.Comparatively, by “asynchronous reentry into the cell cycle” is meantthat the healthy cells, for example HSPCs, in G1 arrest due to theeffect of a CDK4/6 inhibitor compound within relatively differentcollective timeframes or at relatively different rates upon dissipationof the compound's effect such as PD0332991.

By “off-cycle” or “drug holiday” is meant a time period during which thesubject is not administered or exposed to a chemotherapeutic. Forexample, in a treatment regime wherein the subject is administered thechemotherapeutic for 21 straight days and is not administered thechemotherapeutic for 7 days, and the regime is repeated a number oftimes, the 7 day period of non-administration is considered the“off-cycle” or “drug holiday.” Off-target and drug holiday may alsorefer to an interruption in a treatment regime wherein the subject isnot administered the chemotherapeutic for a time due to a deleteriousside effect, for example, myelosuppression.

The subject treated is typically a human subject, although it is to beunderstood the methods described herein are effective with respect toother animals, such as mammals and vertebrate species. Moreparticularly, the term subject can include animals used in assays suchas those used in preclinical testing including but not limited to mice,rats, monkeys, dogs, pigs and rabbits; as well as domesticated swine(pigs and hogs), ruminants, equine, poultry, felines, bovines, murines,canines, and the like.

Active Compounds

In one embodiment, the invention is directed to compounds or the use ofsuch compounds of Formula I, II, III, IV, or V:

or a pharmaceutically acceptable salt thereof;wherein:Z is —(CH₂)_(x)— wherein x is 1, 2, 3 or 4 or —O—(CH₂)_(z)— wherein z is2, 3 or 4;each X is independently CH or N;each X′ is independently, CH or N;X″ is independently CH₂, S or NH, arranged such that the moiety is astable 5-membered ring; R, R⁸, and R¹¹ are independently H, C₁-C₃ alkylor haloalkyl, cycloalkyl or cycloalkyl containing one or moreheteroatoms selected from N, O or S; -(alkylene)m-C₃-C₈ cycloalkyl,-(alkylene)_(m)-aryl, -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(0)-NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(0)n-R⁵, or -(alkylene)_(m)-S(0)n-NR³R⁴ any of whichmay be optionally independently substituted with one or more R groups asallowed by valance, and wherein two R^(x) groups bound to the same oradjacent atoms may optionally combine to form a ring;each R¹ is independently aryl, alkyl, cycloalkyl or haloalkyl, whereineach of said alkyl, cycloalkyl and haloalkyl groups optionally includesO or N heteroatoms in place of a carbon in the chain and two R¹'s onadjacent ring atoms or on the same ring atom together with the ringatom(s) to which they are attached optionally form a 3-8-membered cycle;y is 0, 1, 2, 3 or 4;R² is -(alkylene)_(m)-heterocyclo, -(alkylene)_(m)-heteroaryl,-(alkylene)_(m)-NR³R⁴, (alkylene)_(m)-C(O)—NR³R⁴;-(alkylene)_(m)-C(O)—O-alkyl; -(alkylene)_(m)-O—R⁵,(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atom may optionally combine to form a ring and whereinm is 0 or 1 and n is 0, 1 or 2;R³ and R⁴ at each occurrence are independently:

-   -   (i) hydrogen or    -   (ii) alkyl, cycloalkyl, heterocyclo, aryl, heteroaryl,        cycloalkylalkyl, heterocycloalkyl, arylalkyl, or heteroarylalkyl        any of which may be optionally independently substituted with        one or more R^(x) groups as allowed by valance, and wherein two        R^(x) groups bound to the same or adjacent atom may optionally        combine to form a ring; or R³ and R⁴ together with the nitrogen        atom to which they are attached may combine to form a        heterocyclo ring optionally independently substituted with one        or more R^(x) groups as allowed by valance, and wherein two        R^(x) groups bound to the same or adjacent atom may optionally        combine to form a ring;        R⁵ and R⁵* at each occurrence is:    -   (i) hydrogen or    -   (ii) alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclo, aryl,        heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or        heteroarylalkyl any of which may be optionally independently        substituted with one or more R^(x) groups as allowed by valance;        R^(x) at each occurrence is independently, halo, cyano, nitro,        oxo, alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, heterocycloalkyl,        -(alkylene)_(m)-OR⁵, -(alkylene)_(m)-O-alkylene-OR⁵,        -(alkylene)_(m)-S(O)_(n)—R⁵, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-CN, -(alkylene)_(m)-C(O)—R⁵,        -(alkylene)_(m)-C(S)—R⁵, -(alkylene)_(m)-C(O)—OR⁵,        -(alkylene)_(m)-O—C(O)—R⁵, -(alkylene)_(m)-C(S)—OR⁵,        -(alkylene)_(m)-C(O)-(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—NR³R⁴,        -(alkylene)_(m)-N(R³)—C(S)—NR³R⁴, -(alkylene)_(m)-N(R³)—C(O)—R⁵,        -(alkylene)_(m)-N(R³)—C(S)—R⁵, -(alkylene)_(m)-O—C(O)—NR³R⁴,        -(alkylene)_(m)-O—C(S)—NR³R⁴, -(alkylene)_(m)-S02-NR³R⁴,        -(alkylene)_(m)-N(R³)—SO₂—R⁵, -(alkylene)_(m)-N(R³)—SO₂—NR³R⁴,        -(alkyl ene)_(m)-N(R³)—C(O)—OR⁵) -(alkylene)_(m)-N(R³)—C(S)—OR⁵,        or -(alkylene)_(m)-N(R³)—SO₂—R⁵; wherein:    -   said alkyl, haloalkyl, alkenyl, alkynyl, cycloalkyl,        cycloalkenyl, heterocyclo, aryl, heteroaryl, arylalkyl,        heteroarylalkyl, cycloalkylalkyl, and heterocycloalkyl groups        may be further independently substituted with one or more        -(alkylene)_(m)-CN, -(alkylene)_(m)-OR⁵,        -(alkylene)_(m)-S(O)_(n-)R⁵*, -(alkylene)_(m)-NR³*R⁴*,        -(alkylene)_(m)-C(O)—R⁵*, -(alkylene)_(m)-C(═S)R⁵*,        -(alkylene)_(m)-C(═O)O R⁵*, -(alkylene)_(m)-OC(═O)R⁵*,        -(alkylene)_(m)-C(S)—OR⁵*, -(alkylene)_(m)-C(O)—NR³*R⁴*,        -(alkylene)_(m)-C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(S)—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—R⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—R⁵*, -(alkylene)_(m)-O—C(O)—NR³*R⁴*,        -(alkylene)_(m)-O—C(S)—NR³*R⁴*, -(alkylene)_(m)-S02-NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,        -(alkylene)_(m)-N(R³*)—SO₂—NR³*R⁴*,        -(alkylene)_(m)-N(R³*)—C(O)—OR⁵*,        -(alkylene)_(m)-N(R³*)—C(S)—OR⁵*, or        -(alkylene)_(m)-N(R³*)—SO₂—R⁵*,    -   n is 0, 1 or 2, and    -   m is 0 or 1;        R³* and R⁴* at each occurrence are independently:    -   (i) hydrogen or    -   (ii) alkyl, alkenyl, alkynyl cycloalkyl, heterocyclo, aryl,        heteroaryl, cycloalkylalkyl, heterocycloalkyl, arylalkyl, or        heteroarylalkyl any of which may be optionally independently        substituted with one or more R^(x) groups as allowed by valance;        or R³* and R⁴* together with the nitrogen atom to which they are        attached may combine to form a heterocyclo ring optionally        independently substituted with one or more R^(x) groups as        allowed by valance; and        R⁶ is H or lower alkyl, -(alkylene)_(m)-heterocyclo,        -(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,        -(alkylene)_(m)-C(0)-NR³R⁴; -(alkylene)_(m)-0-R⁵,        -(alkylene)_(m)-S(0)n-R⁵, or -(alkylene)_(m)-S(0)_(n)-NR³R⁴ any        of which may be optionally independently substituted with one or        more R^(x) groups as allowed by valance, and wherein two R^(x)        groups bound to the same or adjacent atoms may optionally        combine to form a ring; and        R¹⁰ is (i) NHR^(A), wherein R^(A) is unsubstituted or        substituted C₁-C₈ alkyl, cycloalkylalkyl, or -TT-RR, C₁-C₈        cycloalkyl or cycloalkyl containing one or more heteroatoms        selected from N, O, and S; TT is an unsubstituted or substituted        C₁-C₈ alkyl or C₃-C₈ cycloalkyl linker; and RR is a hydroxyl,        unsubstituted or substituted C₁-C₆ alkoxy, amino, unsubstituted        or substituted C₁-C₆ alkylamino, unsubstituted or substituted        di-C₁-C₆ alkylamino, unsubstituted or substituted C₆-C₁₀ aryl,        unsubstituted or substituted heteroaryl comprising one or two 5-        or 6-member rings and 1-4 heteroatoms selected from N, O and S,        unsubstituted or substituted C₃-C₁₀ carbocycle, or unsubstituted        or substituted heterocycle comprising one or two 5- or 6-member        rings and 1-4 heteroatoms selected from N, O and S; or (ii)        —C(O)—R¹² or —C(O)O—R¹³, wherein R¹² is NHR^(A) or R^(A) and R¹³        is R^(A);        or a pharmaceutically acceptable salt, prodrug or isotopic        variant, for example, partially or fully deuterated form        thereof.

In some aspects, the compound is of Formula I or Formula II and R⁶ isabsent.

In some aspects, the compound is of Formula III:

and the variables are as defined for compounds of Formulae I and II andpharmaceutically acceptable salts thereof.

In some aspects, R^(x) is not further substituted.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-heteroaryl, -(alkylene)_(m)-NR³R⁴,-(alkylene)_(m)-C(O)—NR³R⁴; -(alkylene)_(m)-O—R⁵,-(alkylene)_(m)-S(O)_(n)—R⁵, or -(alkylene)_(m)-S(O)_(n)—NR³R⁴ any ofwhich may be optionally independently substituted with one or more R^(x)groups as allowed by valance, and wherein two R^(x) groups bound to thesame or adjacent atom may optionally combine to form a ring and whereinm is 0 or 1 and n is 0, 1 or 2.

In some aspects, R⁸ is hydrogen or C₁-C₃ alkyl.

In some aspects, R is hydrogen or C₁-C₃ alkyl.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴,-(alkylene)_(m)-C(O)—O-alkyl or -(alkylene)_(m)-OR⁵ any of which may beoptionally independently substituted with one or more R^(x) groups asallowed by valance, and wherein two R^(x) groups bound to the same oradjacent atom may optionally combine to form a ring.

In some aspects, R² is -(alkylene)_(m)-heterocyclo,-(alkylene)_(m)-NR³R⁴, -(alkylene)_(m)-C(O)—NR³R⁴,-(alkylene)_(m)-C(O)—O-alkyl or -(alkylene)_(m)-OR⁵ without furthersubstitution.

In some aspects, m in R² is 1. In a further aspect the alkylene in R² ismethylene.

In some aspects, R² is

wherein:R²* is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-,-(alkylene)_(m)-C(O)-(alkylene)_(m)-,-(alkylene)_(m)-S(O)₂-(alkylene)_(m)- and-(alkylene)_(m)-NH-(alkylene)_(m)-wherein each m is independently 0 or1;P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group;each R^(x1) is independently-(alkylene)_(m)-(C(O))_(m)-(alkylene)_(m)-(N(R^(N)))_(m)-(alkyl)_(m)wherein each m is independently 0 or 1 provided at least one m is 1,—(C(O))—O-alkyl, -(alkylene)_(m)-cycloalkyl wherein m is 0 or 1,—N(R^(N))-cycloalkyl, —C(O)-cycloalkyl, -(alkylene)_(m)-heterocyclylwherein m is 0 or 1, or —N(R^(N))-heterocyclyl, —C(O)-heterocyclyl,—S(O)₂-(alkylene)_(m) wherein m is 1 or 2, wherein:

R^(N) is H, C₁ to C₄ alkyl or C₁ to C₆ heteroalkyl, and

wherein two R^(x1) can, together with the atoms to which they attach onP, which may be the

same atom, form a ring; and

t is 0, 1 or 2.

In some aspects, each R^(x1) is only optionally substituted byunsubstituted alkyl, halogen or hydroxy.

In some aspects, R^(x1) is hydrogen or unsubstituted C₁-C₄ alkyl.

In some aspects, at least one R^(x1) is -(alkylene)_(m)-heterocyclylwherein m is 0 or 1.

In some aspects, R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some aspects, R² is

In some aspects, R² is

In some aspects, R² is

wherein:R²* is a bond, alkylene, -(alkylene)_(m)-O-(alkylene)_(m)-,-(alkylene)_(m)-C(O)-(alkylene)_(m)-,-(alkylene)_(m)-S(O)₂-(alkylene)_(m)- and-(alkylene)_(m)-NH-(alkylene)_(m)-wherein each m is independently 0 or1;P is a 4- to 8-membered mono- or bicyclic saturated heterocyclyl group;P1 is a 4- to 6-membered monocyclic saturated heterocyclyl group;each R^(x2) is independently hydrogen or alkyl; ands is 0, 1 or 2.

In some aspects, R² is

In some aspects, P1 includes at least one nitrogen.

In some aspects, any alkylene in R²* in any previous aspect is notfurther substituted.

In some aspects, R² is selected from the structures depicted in FIGS.13-15.

In some aspects, R² is

In some aspects, the compound has general Formula I and morespecifically one of the general structures in FIGS. 16-20 wherein thevariables are as previously defined.

In some aspects, the compound has general Formula Ia:

wherein R¹, R², R and y are as previously defined.

In some embodiments, the compound has Formula Ia and R is alkyl.

In some embodiments, the compound has Formula Ia and R is H.

In some embodiments, the compound has Formula Ia and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ia and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or unsubstituted C₁-C₄ alkyl andR²* is as previously defined.

In some embodiments, the compound has Formula Ib:

wherein R² and R are as previously defined.

In some embodiments, the compound has Formula Ib and R is alkyl.

In some embodiments, the compound has Formula Ib and R is H.

In some embodiments, the compound has Formula Ib and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ib and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ic:

wherein R² and R are as previously defined.

In some embodiments, the compound has Formula Ic and R is alkyl.

In some embodiments, the compound has Formula Ic and R is H.

In some embodiments, the compound has Formula Ic and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ic and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Id:

wherein R² and R are as previously defined.

In some embodiments, the compound has Formula Id and R is alkyl.

In some embodiments, the compound has Formula Id and R is H.

In some embodiments, the compound has Formula Id and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Id and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ie:

In some embodiments, the compound has Formula Ie and R is alkyl.

In some embodiments, the compound has Formula Ie and R is H.

In some embodiments, the compound has Formula Ie and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ie and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula If:

In some embodiments, the compound has Formula If and R is alkyl.

In some embodiments, the compound has Formula If and R is H.

In some embodiments, the compound has Formula If and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula If and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ig:

In some embodiments, the compound has Formula Ig and R is alkyl.

In some embodiments, the compound has Formula Ig and R is H.

In some embodiments, the compound has Formula Ig and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ig and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ih:

In some embodiments, the compound has Formula Ih and R is alkyl.

In some embodiments, the compound has Formula Ih and R is H.

In some embodiments, the compound has Formula Ih and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ih and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ii:

In some embodiments, the compound has Formula Ii and R is alkyl.

In some embodiments, the compound has Formula Ii and R is H.

In some embodiments, the compound has Formula Ii and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group and R²*, R^(x1) and t are as previously defined.

In some embodiments, the compound has Formula Ii and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl and R²* is aspreviously defined.

In some embodiments, the compound has Formula Ij:

In some embodiments, the compound has Formula Ij and R is alkyl.

In some embodiments, the compound has Formula Ij and R is H.

In some embodiments, the compound has Formula Ij and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Ij and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Ij and R is H, and both Xare N.

In some embodiments, the compound has the structure:

In some embodiments, the compound has Formula Ik and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Ik and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Il:

In some embodiments, the compound has Formula II and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Il and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula Im:

In some embodiments, the compound has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula IIa:

In some embodiments, the compound has Formula IIa and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula IIa and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some embodiments, the compound has Formula IIb:

In some embodiments, the compound has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group.

In some embodiments, the compound has Formula Im and R² is

wherein P* is a 4- to 8-membered mono- or bicyclic saturatedheterocyclyl group, R^(x1) is hydrogen or C₁-C₄ alkyl.

In some aspects, the active compound is:

Further specific compounds that fall within the present invention andthat can be used in the disclosed methods of treatment and compositionsinclude the structures listed in Table 1 below.

TABLE 1 Structures of Anti-Neoplastic and Anti-Proliferative AgentsStructure Reference Structure A

B

C

D

E

F

G

H

I

J

K

L

M

N

O

P

Q

R

S

T

U

V

W

X

Y

Z

AA

BB

CC

DD

EE

FF

GG

HH

II

JJ

KK

LL

MM

NN

OO

PP

QQ

RR

SS

TT

UU

VV

WW

XX

YY

ZZ

AAA

BBB

CCC

DDD

EEE

FFF

GGG

HHH

III

JJJ

KKK

LLL

MMM

NNN

OOO

PPP

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Isotopic Substitution

The present invention includes compounds and the use of compounds withdesired isotopic substitutions of atoms, at amounts above the naturalabundance of the isotope, i.e., enriched. Isotopes are atoms having thesame atomic number but different mass numbers, i.e., the same number ofprotons but a different number of neutrons. By way of general exampleand without limitation, isotopes of hydrogen, for example, deuterium(²H) and tritium (³H) may be used anywhere in described structures.Alternatively or in addition, isotopes of carbon, e.g., ¹³C and ¹⁴C, maybe used. A preferred isotopic substitution is deuterium for hydrogen atone or more locations on the molecule to improve the performance of thedrug. The deuterium can be bound in a location of bond breakage duringmetabolism (an α-deuterium kinetic isotope effect) or next to or nearthe site of bond breakage (a β-deuterium kinetic isotope effect).

Substitution with isotopes such as deuterium can afford certaintherapeutic advantages resulting from greater metabolic stability, suchas, for example, increased in vivo half-life or reduced dosagerequirements. Substitution of deuterium for hydrogen at a site ofmetabolic break down can reduce the rate of or eliminate the metabolismat that bond. At any position of the compound that a hydrogen atom maybe present, the hydrogen atom can be any isotope of hydrogen, includingprotium (¹H), deuterium (²H) and tritium (³H). Thus, reference herein toa compound encompasses all potential isotopic forms unless the contextclearly dictates otherwise.

The term “isotopically-labeled” analog refers to an analog that is a“deuterated analog”, a “¹³C-labeled analog,” or a“deuterated/¹³C-labeled analog.” The term “deuterated analog” means acompound described herein, whereby a H-isotope, i.e., hydrogen/protium(¹H), is substituted by a H-isotope, i.e., deuterium (²H). Deuteriumsubstitution can be partial or complete. Partial deuterium substitutionmeans that at least one hydrogen is substituted by at least onedeuterium. In certain embodiments, the isotope is 90, 95 or 99% or moreenriched in an isotope at any location of interest. In some embodimentsit is deuterium that is 90, 95 or 99% enriched at a desired location.

Further specific compounds that fall within the present invention andthat can be used in the disclosed methods of treatment and compositionsinclude the structures of Formula I, II, III, IV, or V listed in Table 1below.

Rb-Positive Cancers and Proliferative Disorders

In particular, the active compounds described herein can be used totreat a subject suffering from a Rb-positive cancer or other Rb-positiveabnormal cellular proliferative disorder. In some embodiments, thecancer or cellular proliferation disorder is a CDK4/6-replicationdependent cancer or cellular proliferation disorder, which refers to acancer or cellular proliferation disorder that requires the activity ofCDK4/6 for replication or proliferation, or which may be growthinhibited through the activity of a selective CDK4/6 inhibitor. Cancersand disorders of such type can be characterized by (e.g., that has cellsthat exhibit) the presence of a functional Retinoblastoma protein. Suchcancers and disorders are classified as being Rb-positive. Rb-positiveabnormal cellular proliferation disorders, and variations of this termas used herein, refer to disorders or diseases caused by uncontrolled orabnormal cellular division which are characterized by the presence of afunctional Retinoblastoma protein, which can include cancers. In oneaspect of the present invention, the compounds and methods describedherein can be used to treat a non-cancerous Rb-positive abnormalcellular proliferation disorder. Examples of such disorders may includenon-malignant lymphoproliferation, non-malignant breast neoplasms,psoriasis, arthritis, dermatitis, pre-cancerous colon lesions or pulps,angiogenesis disorders, immune mediated and non-immune mediatedinflammatory diseases, arthritis, age-related macular degeneration,diabetes, and other non-cancerous or benign cellular proliferationdisorders.

Targeted cancers suitable for administration of a compound describedherein may include Rb-positive: estrogen-receptor positive,HER2-negative advanced breast cancer, late-line metastatic breastcancer, liposarcoma, non-small cell lung cancer, liver cancer, ovariancancer, glioblastoma, refractory solid tumors, retinoblastoma positivebreast cancer as well as retinoblastoma positive endometrial, vaginaland ovarian cancers and lung and bronchial cancers, adenocarcinoma ofthe colon, adenocarcinoma of the rectum, central nervous system germcell tumors, teratomas, estrogen receptor-negative breast cancer,estrogen receptor-positive breast cancer, familial testicular germ celltumors, HER2-negative breast cancer, HER2-positive breast cancer, malebreast cancer, ovarian immature teratomas, ovarian mature teratoma,ovarian monodermal and highly specialized teratomas, progesteronereceptor-negative breast cancer, progesterone receptor-positive breastcancer, recurrent breast cancer, recurrent colon cancer, recurrentextragonadal germ cell tumors, recurrent extragonadal non-seminomatousgerm cell tumor, recurrent extragonadal seminomas, recurrent malignanttesticular germ cell tumors, recurrent melanomas, recurrent ovarian germcell tumors, recurrent rectal cancer, stage III extragonadalnon-seminomatous germ cell tumors, stage III extragonadal seminomas,stage III malignant testicular germ cell tumors, stage III ovarian germcell tumors, stage IV breast cancers, stage IV colon cancers, stage IVextragonadal non-seminomatous germ cell tumors, stage IV extragonadalseminoma, stage IV melanomas, stage IV ovarian germ cell tumors, stageIV rectal cancers, testicular immature teratomas, testicular matureteratomas. In particular embodiments, the targeted cancers includedestrogen-receptor positive, HER2-negative advanced breast cancer,late-line metastatic breast cancer, liposarcoma, non-small cell lungcancer, liver cancer, ovarian cancer, glioblastoma, refractory solidtumors, retinoblastoma positive breast cancer as well as retinoblastomapositive endometrial, vaginal and ovarian cancers and lung and bronchialcancers, metastatic colorectal cancer, metastatic melanoma with CDK4mutation or amplification, or cisplatin-refractory, unresectable germcell tumors.

In one embodiment, the Rb-positive cancer is selected from anRb-positive carcinoma, sarcoma, including, but not limited to, lungcancer, bone cancer, pancreatic cancer, skin cancer, cancer of the heador neck, cutaneous or intraocular melanoma, uterine cancer, ovariancancer, rectal cancer, cancer of the anal region, stomach cancer, coloncancer, breast cancer, uterine cancer, carcinoma of the fallopian tubes,carcinoma of the endometrium, carcinoma of the cervix, carcinoma of thevagina, carcinoma of the vulva, cancer of the esophagus, cancer of thesmall intestine, cancer of the endocrine system, cancer of the thyroidgland, cancer of the parathyroid gland, cancer of the adrenal gland,sarcoma of soft tissue, cancer of the urethra, cancer of the penis,prostate cancer, cancer of the bladder, cancer of the kidney or ureter,renal cell carcinoma, carcinoma of the renal pelvis, neoplasms of thecentral nervous system (CNS), primary CNS lymphoma, spinal axis tumors,brain stem glioma, pituitary adenoma, or a combination of one or more ofthe foregoing cancers.

In one embodiment, the Rb-positive cancer is selected from the groupconsisting of Rb-positive: fibrosarcoma, myxosarcoma, chondrosarcoma,osteosarcoma, chordoma, malignant fibrous histiocytoma, hemangiosarcoma,angiosarcoma, lymphangiosarcoma. Mesothelioma, leiomyosarcoma,rhabdomyosarcoma, squamous cell carcinoma; epidermoid carcinoma,malignant skin adnexal tumors, adenocarcinoma, hepatoma, hepatocellularcarcinoma, renal cell carcinoma, hypernephroma, cholangiocarcinoma,transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cellcarcinoma, glioma anaplastic; glioblastoma multiforme, neuroblastoma,medulloblastoma, malignant meningioma, malignant schwannoma,neurofibrosarcoma, parathyroid carcinoma, medullary carcinoma ofthyroid, bronchial carcinoid, pheochromocytoma, Islet cell carcinoma,malignant carcinoid, malignant paraganglioma, melanoma, Merkel cellneoplasm, cystosarcoma phylloide, salivary cancers, thymic carcinomas,bladder cancer, and Wilms tumor.

The presence or normal functioning of the retinoblastoma (Rb) tumorsuppressor protein (Rb-positive) can be determined through any of thestandard assays known to one of ordinary skill in the art, including butnot limited to Western Blot, ELISA (enzyme linked immunoadsorbentassay), IHC (immunohistochemistry), and FACS (fluorescent activated cellsorting). The selection of the assay will depend upon the tissue, cellline or surrogate tissue sample that is utilized e.g., for exampleWestern Blot and ELISA may be used with any or all types of tissues,cell lines or surrogate tissues, whereas the IHC method would be moreappropriate wherein the tissue utilized in the methods of the presentinvention was a tumor biopsy. FACs analysis would be most applicable tosamples that were single cell suspensions such as cell lines andisolated peripheral blood mononuclear cells. See for example, US20070212736 “Functional Immunohistochemical Cell Cycle Analysis as aPrognostic Indicator for Cancer”. Alternatively, molecular genetictesting may be used for determination of retinoblastoma gene status.Molecular genetic testing for retinoblastoma includes the following asdescribed in Lohmann and Gallie “Retinoblastoma. Gene Reviews” (2010)http://www.ncbi.nlm.nih.gov/bookshelf/br.fcgi?book=gene&part=retinoblastomaor Parsam et al. “A comprehensive, sensitive and economical approach forthe detection of mutations in the RB1 gene in retinoblastoma” Journal ofGenetics, 88(4), 517-527 (2009).

In some embodiments, the cancer to be treated is selected fromestrogen-receptor positive, HER2-negative advanced breast cancer,late-line metastatic breast cancer, liposarcoma, non-small cell lungcancer, liver cancer, ovarian cancer, glioblastoma, refractory solidtumors, retinoblastoma positive breast cancer as well as retinoblastomapositive endometrial, vaginal and ovarian cancers and lung and bronchialcancers.

CDK-Replication Dependent Cells and Cyclin-Dependent Kinase Inhibitors

Tissue-specific stem cells and subsets of other resident proliferatingcells are capable of self-renewal, meaning that they are capable ofreplacing themselves throughout the adult mammalian lifespan throughregulated replication. Additionally, stem cells divide asymmetrically toproduce “progeny” or “progenitor” cells that in turn produce variouscomponents of a given organ. For example, in the hematopoietic system,the hematopoietic stem cells give rise to progenitor cells which in turngive rise to all the differentiated components of blood (e.g., whiteblood cells, red blood cells, and platelets) (see FIG. 1).

It has been found that certain proliferating cells, such as HSPCs,require the enzymatic activity of the proliferative kinasescyclin-dependent kinase 4 (CDK4) and/or cyclin-dependent kinase 6 (CDK6)for cellular replication. In contrast, the majority of proliferatingcells in adult mammals (e.g., the more differentiated blood-formingcells in the bone marrow) do not require the activity of CDK4 and/orCDK6 (i.e., CDK4/6). These differentiated cells can proliferate in theabsence of CDK4/6 activity by using other proliferative kinases, such ascyclin-dependent kinase 2 (CDK2) or cyclin-dependent kinase 1 (CDK1).

The present invention includes methods of treating certain cancers, inparticular Rb-positive cancers, while minimizing the deleterious effectson CDK4/6-replication dependent healthy cells in a subject, and inparticular, hematopoietic cells and/or progenitor cells (HSPCs), by theadministration of a compound described herein to treat a specificRb-positive cancer.

In one embodiment, the use of a compound described herein as achemotherapeutic allows for an accelerated hematological recovery andreduced hematological deficiency risk due to HSPC replication delaycompared to the use of other CDK4/6 inhibitors, for example, PD0332991.In one embodiment, the use of a compound described herein as achemotherapeutic allows for a reduced or minimized off-cycle or drugholiday during the course of treatment compared to current treatmentmodalities using other CDK4/6 inhibitors, for example PD0332991. In oneembodiment, the use of the compounds described herein aschemotherapeutics allows for the elimination of an off-cycle or drugholiday. In one embodiment, the use of the compounds described herein aschemotherapeutics allows for an extended period of administration withfewer off-cycle days or drug holidays compared to the use of currenttreatment modalities using other CDK4/6 inhibitors, for examplePD0332991. In one embodiment, the use of the compounds described hereinas chemotherapeutics allows for a faster blood count recovery during anoff-cycle or drug holiday than the use of current modalities using otherCDK4/6 inhibitors, for example PD0332991.

In certain embodiments, the compound administered is selected from thegroup consisting of a compound or composition comprising Formula I,Formula II, Formula III, Formula IV, or Formula V, or a combinationthereof. In certain embodiments, the compound administered is selectedfrom the group consisting of a compound selected from Table 1.

In certain aspects, compounds, methods, and compositions are provided aschemotherapeutics which reduce or limit the deleterious effect of CDK4/6inhibition on CDK4/6-replication dependent healthy cells in a subjectundergoing CDK4/6 inhibitory treatment for a Rb-positive cancer, themethod comprising administering an effective amount of a compounddescribed herein, wherein a substantial portion of theCDK4/6-replication dependent healthy cells return to pre-treatmentbaseline cell cycle activity (i.e., reenter the cell-cycle) within lessthan about 24, 30, 36, or 40 hours of administration of the compound. Incertain embodiments wherein the compound has an IC₅₀ CDK4 inhibitoryconcentration that is at least 1500 times less than its IC₅₀ inhibitoryconcentration for CDK2. In certain embodiments, the compoundadministered is selected from the group consisting of the compound or acomposition comprising Formula I, Formula II, Formula III, Formula IV,or Formula V, or a pharmaceutically acceptable composition, salt,isotopic analog, or prodrug thereof. In certain embodiments, thecompound administered is selected from a compound contained in Table 1,or a pharmaceutically acceptable composition, salt, isotopic analog, orprodrug thereof. In one embodiment, the CDK4/6-replication dependentcells are hematopoietic stem cells and/or progenitor cells (HSPCs).

In certain aspects, compounds, methods, and composition are provided foruse as chemotherapeutics which limit the deleterious effect of CDK4/6inhibition on CDK4/6-replication dependent healthy cells in a subjectundergoing treatment for a Rb-positive cancer, the method comprisingadministering an effective amount of a compound described herein,wherein a substantial portion of the CDK4/6-replication dependenthealthy cells synchronously reenter the cell-cycle within less thanabout 24, 30, 36 or 40 hours following the dissipation of the compound'sinhibitory effect. In certain embodiments wherein the compound has anIC₅₀ CDK4 inhibitory concentration that is at least 1500 times less thanits IC₅₀ inhibitory concentration for CDK2. In certain embodiments, thecompound administered is selected from the group consisting of thecompound or a composition comprising Formula I, Formula II, Formula III,Formula IV, or Formula V, or a pharmaceutically acceptable composition,salt, isotopic analog, or prodrug thereof. In certain embodiments, thecompound administered is selected from a compound contained in Table 1,or a pharmaceutically acceptable composition, salt, isotopic analog, orprodrug thereof. In one embodiment, the CDK4/6-replication dependentcells are hematopoietic stem cells and/or progenitor cells (HSPCs).

In certain aspects, compounds, methods, and composition are provided foruse as chemotherapeutics which limit the deleterious effect of CDK4/6inhibition on CDK4/6-replication dependent healthy cells in a subject,the method comprising administering an effective amount of a compounddescribed herein to a subject with a Rb-positive cancer, wherein asubstantial portion of the CDK4/6-replication dependent healthy cellsreenter the cell-cycle synchronously within less than about 24, 30, 36,or 40 hours following the dissipation of the compound's CDK4/6inhibitory effect. In one embodiment, the administered compound has anIC₅₀ CDK4 inhibitory concentration that is more than 500 times less thanits IC₅₀ inhibitory concentration for CDK2. In certain embodiments, asubstantial portion of the CDK4/6-replication dependent healthy cellsreenter the cell-cycle synchronously within less than about 24, 30, 36,or 40 hours from the point in which the compound's concentration levelin the subject's blood drops below a therapeutic effectiveconcentration. In certain embodiments, the compound administered isselected from the group consisting of the compound or a compositioncomprising Formula I, Formula II, Formula III, Formula IV, or Formula V,or a pharmaceutically acceptable composition, salt, isotopic analog, orprodrug thereof. In certain embodiments, the compound administered isselected from a compound contained in Table 1, or a pharmaceuticallyacceptable composition, salt, isotopic analog, or prodrug thereof. Inone embodiment, the CDK4/6-replication dependent cells are hematopoieticstem cells and/or progenitor cells (HSPCs). In one embodiment theCDK4/6-replication dependent healthy cells are renal epithelial cells.

In certain embodiments, the compound administered is selected from thegroup consisting of the compound or a composition comprising Formula I,Formula II, Formula III, Formula IV, or Formula V, or a pharmaceuticallyacceptable composition, salt, isotopic analog, or prodrug thereof, orcompound contained in Table 1, or a pharmaceutically acceptablecomposition, salt, isotopic analog, or prodrug thereof, wherein theeffect of the compound is short term and transient in nature, allowing asignificant portion of the CDK4/6-replication dependent healthy cells tosynchronously renter the cell-cycle quickly, for example within lessthan about 24, 30, 36, or 40 hours of the last administration of thecompound.

The compounds for use in the described methods are highly selective,potent CDK4/6 inhibitors, with minimal CDK2 inhibitory activity. In oneembodiment, a compound for use in the methods described herein has aCDK4/CycD1 IC₅₀ inhibitory concentration value that is >1500times, >1800 times, >2000 times, >2200 times, >2500 times, >2700times, >3000 times, >3200 times or greater lower than its respectiveIC₅₀ concentration value for CDK2/CycE inhibition. In one embodiment, acompound for use in the methods described herein has an IC₅₀concentration value for CDK4/CycD1 inhibition that is about <1.50 nM,<1.25 nM, <1.0 nM, <0.90 nM, <0.85 nM, <0.80 nM, <0.75 nM, <0.70 nM,<0.65 nM, <0.60 nM, <0.55 nM, or less. In one embodiment, a CDK4/6inhibitor for use in the methods described herein has an IC₅₀concentration value for CDK2/CycE inhibition that is about >1.0μM, >1.25 μM, >1.50 μM, >1.75 μM, >2.0 μM, >2.25 μM, >2.50 μM, >2.75μM, >3.0 μM, >3.25 μM, >3.5 μM or greater. In one embodiment, a compoundfor use in the methods described herein has an IC₅₀ concentration valuefor CDK2/CycA IC₅₀ that is >0.80 μM, >0.85 μM, >0.90 μM, >0.95μM, >0.1.0 μM, >1.25 μM, >1.50 μM, >1.75 μM, >2.0 μM, >2.25 μM, >2.50μM, >2.75 uM, >3.0 μM or greater.

In certain embodiments, the compounds useful in the described methodsmay provide for a transient and quickly reversible G1-arrest ofCDK4/6-replication dependent healthy cells while providing for growthinhibition of CDK4/6-replication dependent cancers. By having alimited-term transient effect, the use of such compounds aschemotherapeutics allows for the faster reentry of theCDK4/6-replication dependent healthy cells into the cell cycle followingcessation of the treatment compared to, for example, longer actingCDK4/6 inhibitors such as PD0332991. The quicker dissipation of the G1arresting effect on CDK4/6-replication dependent healthy cells makessuch compounds preferable over longer acting CDK4/6 inhibitors insituations where: 1) the subject will be exposed to closely spacedtreatments, wherein the use of a longer acting CDK4/6 inhibitor wouldprohibit the cycling of the CDK4/6-replication dependent healthy cellsbetween exposures; 2) continuous or long-term treatment regimens whereinthe long-term G1 arrest of CDK4/6-replication dependent healthy cells isa side effect of growth inhibition of the targeted cancer, and thesubject would benefit from the CDK4/6-replication dependent healthycells quickly reentering the cell-cycle following cessation of thetreatment regime, between dosing of the inhibitor in a continuousregime, or between breaks in treatment in order to limit replicationdelay, thus reducing, limiting, or ameliorating further healthy celldamage, for example bone marrow suppression, upon cessation of thetreatment. According to the present invention, chemotherapeutic regimenswith the selective compounds described herein can be achieved by anumber of different dosing schedules, including on-cycle/off-cycleregimes and continuous treatment regimes.

In one embodiment, the compounds described herein are used inCDK4/6-replication dependent healthy cell cycling strategies wherein asubject is exposed to regular, repeated chemotherapeutic treatments foran Rb-positive cancer. Such cycling allows CDK4/6-replication dependentcells to regenerate damaged blood cell lineages between regular,repeated treatments, and reduces the risk associated with long termCDK4/6 inhibition. This cycling between a state of G1-arrest and a stateof replication is not feasible in limited time-spaced, repeated agentexposures using longer acting CDK4/6 inhibitors such as PD0332991, asthe lingering G1-arresting effect of the compound prohibit significantand meaningful reentry into the cell-cycle by the CDK4/6-replicationdependent cells before the next exposure to the CDK4/6 inhibitor, ordelay the healthy cells from entering the cell cycle and reconstitutingdamaged tissues or cells following treatment cessation.

In one embodiment, the use of a compound described herein provides for arapid, reentry into the cell cycle by CDK4/6-replication dependenthealthy cells, for example HSPCs, so that the cells return topre-treatment baseline cell cycle activity within less than about 40hours, 36 hours, 30 hours, 28 hours, 24 hours or less. In oneembodiment, the use of a compound described herein provides for a rapid,reentry into the cell cycle by CDK4/6-replication dependent healthycells, for example HSPCs, so that the cells approach pre-treatmentbaseline cell cycle activity within less than about 40 hours, 36 hours,30 hours, 28 hours, 24 hours, 18 hours, 16 hours, 14 hours, 12 hours orless. In one embodiment, the use of a compound described herein providesfor a rapid, reentry into the cell cycle by CDK4/6-replication dependentcells so that the cells return to pre-treatment baseline cell cycleactivity within less than about 40 hours, 36 hours, 30 hours, 28 hours,24 hours, 18 hours, 16 hours, 14 hours, 12 hours or less from the lastadministration of a compound described herein. In one embodiment, theuse of a compound described herein provides for a rapid, reentry intothe cell cycle by CDK4/6-replication dependent healthy cells so that thecells approach pre-treatment baseline cell cycle activity within lessthan about 40 hours, 36 hours, 30 hours, 28 hours, 24 hours, 18 hours,16 hours, 14 hours, 12 hours or less from the last administration of thecompound. In one embodiment, the use of a compound described hereinprovides for a rapid, reentry into the cell cycle by CDK4/6-replicationdependent healthy cells so that the cells approach pre-treatmentbaseline cell cycle activity within less than about 40 hours, 36 hours,30 hours, 28 hours, 24 hours, 18 hours, 16 hours, 14 hours, 12 hours orless from the point in which the compound's concentration level in thesubject's blood drops below a therapeutic effective concentration. Inone embodiment, the CDK4/6-replication dependent healthy cells areHSPCs. In one embodiment, the CDK4/6-replication dependent healthy cellsare renal epithelial cells. In one embodiment, the rapid reentry intothe cell-cycle is synchronous.

In one embodiment, the use of a compound described herein provides for arapid, reentry into the cell cycle by CDK4/6-replication dependenthealthy cells, for example HSPCs, so that a portion of the cells exhibita level of cell cycle activity or are capable of entering the cell cycleand proliferate during a continuous treatment regime, for example, atreatment regime wherein the compound is administered for an extendedperiod, for example, 5 continuous days, 7 continuous days, 10 continuousdays, 14 continuous days, 18 continuous days, 21 continuous days, 24continuous days, 28 continuous days, 35 continuous days or more. In oneembodiment, a compound useful in a described method is administered fora continuous period, for example, 21, 28, 35 days or more, without therequirement for an off-cycle period or drug holiday. In one embodiment,the use of a compound described herein eliminates the need for anoff-cycle period, drug holiday, or reduction in co-administeredanti-neoplastic compound concentration during treatment.

According to the present invention, a compound described herein can beadministered as a chemotherapeutic to a subject having an Rb-positiveproliferation disorder on any treatment schedule and in any doseconsistent with the prescribed course of treatment. For instance thecompound can be administered once a day, twice a day or three times aday. The compound can be administered on alternating days, or everythird day, or every fourth day, or every fifth day, or every sixth dayor once a week. The compound can be administered every other week ormonthly.

Combination Therapy

In one aspect of the invention, the compounds disclosed herein can bebeneficially administered in combination with another therapeuticregimen for beneficial, additive or synergistic effect.

In one embodiment, a compound/method of the present invention is used incombination with another therapy to treat the Rb-positive cancer. Thesecond therapy can be an immunotherapy. As discussed in more detailbelow, the compound can be conjugated to an antibody, radioactive agent,or other targeting agent that directs the compound to the diseased orabnormally proliferating cell. In another embodiment, the compound isused in combination with another pharmaceutical or a biologic agent (forexample an antibody) to increase the efficacy of treatment with acombined or a synergistic approach. In an embodiment, the compound canbe used with T-cell vaccination, which typically involves immunizationwith inactivated autoreactive T cells to eliminate an Rb-positive cancercell population as described herein. In another embodiment, the compoundis used in combination with a bispecific T-cell Engager (BiTE), which isan antibody designed to simultaneously bind to specific antigens onendogenous T cells and Rb-positive cancer cells as described herein,linking the two types of cells.

In one embodiment, the additional therapy is a monoclonal antibody(MAb). Some MAbs stimulate an immune response that destroys cancercells. Similar to the antibodies produced naturally by B cells, theseMAbs “coat” the cancer cell surface, triggering its destruction by theimmune system. For example, bevacizumab targets vascular endothelialgrowth factor (VEGF), a protein secreted by tumor cells and other cellsin the tumor's microenvironment that promotes the development of tumorblood vessels. When bound to bevacizumab, VEGF cannot interact with itscellular receptor, preventing the signaling that leads to the growth ofnew blood vessels. Similarly, cetuximab and panitumumab target theepidermal growth factor receptor (EGFR), and trastuzumab targets thehuman epidermal growth factor receptor 2 (HER-2). MAbs that bind to cellsurface growth factor receptors prevent the targeted receptors fromsending their normal growth-promoting signals. They may also triggerapoptosis and activate the immune system to destroy tumor cells.

Another group of cancer therapeutic MAbs are the immunoconjugates. TheseMAbs, which are sometimes called immunotoxins or antibody-drugconjugates, consist of an antibody attached to a cell-killing substance,such as a plant or bacterial toxin, a chemotherapy drug, or aradioactive molecule. The antibody latches onto its specific antigen onthe surface of a cancer cell, and the cell-killing substance is taken upby the cell. FDA-approved conjugated MAbs that work this way includeado-trastuzumab emtansine, which targets the HER-2 molecule to deliverthe drug DM1, which inhibits cell proliferation, to HER-2 expressingmetastatic breast cancer cells.

Immunotherapies with T cells engineered to recognize cancer cells viabispecific antibodies (bsAbs) or chimeric antigen receptors (CARs) areapproaches with potential to ablate both dividing and non/slow-dividingsubpopulations of cancer cells.

Bispecific antibodies, by simultaneously recognizing target antigen andan activating receptor on the surface of an immune effector cell, offeran opportunity to redirect immune effector cells to kill cancer cells.The other approach is the generation of chimeric antigen receptors byfusing extracellular antibodies to intracellular signaling domains.Chimeric antigen receptor-engineered T cells are able to specificallykill tumor cells in a MHC-independent way.

In some embodiments, the compound can be administered to the subject incombination with other chemotherapeutic agents. If convenient, thecompounds described herein can be administered at the same time asanother chemotherapeutic agent, in order to simplify the treatmentregimen. In some embodiments, the compound and the otherchemotherapeutic can be provided in a single formulation. In oneembodiment, the use of the compounds described herein is combined in atherapeutic regime with other agents. Such agents may include, but arenot limited to, tamoxifen, midazolam, letrozole, bortezomib,anastrozole, goserelin, an mTOR inhibitor, a PI3 kinase inhibitors, dualmTOR-PI3K inhibitors, MEK inhibitors, RAS inhibitors, ALK inhibitors,HSP inhibitors (for example, HSP70 and HSP 90 inhibitors, or acombination thereof), BCL-2 inhibitors, apopototic inducing compounds,AKT inhibitors, including but not limited to, MK-2206, GSK690693,Perifosine, (KRX-0401), GDC-0068, Triciribine, AZD5363, Honokiol,PF-04691502, and Miltefosine, PD-1 inhibitors including but not limitedto, Nivolumab, CT-011, MK-3475, BMS936558, and AMP-514 or FLT-3inhibitors, including but not limited to, P406, Dovitinib, Quizartinib(AC220), Amuvatinib (MP-470), Tandutinib (MLN518), ENMD-2076, andKW-2449, or combinations thereof. Examples of mTOR inhibitors includebut are not limited to rapamycin and its analogs, everolimus (Afinitor),temsirolimus, ridaforolimus, sirolimus, and deforolimus. Examples of P13kinase inhibitors include but are not limited to Wortmannin,demethoxyviridin, perifosine, idelalisib, PX-866, IPI-145, BAY 80-6946,BEZ235, RP6503, TGR 1202 (RP5264), MLN1117 (INK1117), Pictilisib,Buparlisib, SAR245408 (XL147), SAR245409 (XL765), Palomid 529, ZSTK474,PWT33597, RP6530, CUDC-907, and AEZS-136. Examples of MEK inhibitorsinclude but are not limited to Tametinib, Selumetinib, MEK162, GDC-0973(XL518), and PD0325901. Examples of RAS inhibitors include but are notlimited to Reolysin and siG12D LODER. Examples of ALK inhibitors includebut are not limited to Crizotinib, AP26113, and LDK378. HSP inhibitorsinclude but are not limited to Geldanamycin or17-N-Allylamino-17-demethoxygeldanamycin (17AAG), and Radicicol. In aparticular embodiment, a compound described herein is administered incombination with letrozole and/or tamoxifen. Other chemotherapeuticagents that can be used in combination with the compounds describedherein include, but are not limited to, chemotherapeutic agents that donot require cell cycle activity for their anti-neoplastic effect.

In one embodiment, a CDK4/6 inhibitor described herein can be combinedwith a chemotherapeutic selected from, but are not limited to, Imatinibmesylate (Gleevac®), Dasatinib (Sprycel®), Nilotinib (Tasigna®),Bosutinib (Bosulif®), Trastuzumab (Herceptin®), Pertuzumab (Perjeta™),Lapatinib (Tykerb®), Gefitinib (Iressa®), Erlotinib (Tarceva®),Cetuximab (Erbitux®), Panitumumab (Vectibix®), Vandetanib (Caprelsa®),Vemurafenib (Zelboraf®), Vorinostat (Zolinza®), Romidepsin (Istodax®),Bexarotene (Tagretin®), Alitretinoin (Panretin®), Tretinoin (Vesanoid®),Carfilizomib (Kyprolis™), Pralatrexate (Folotyn®), Bevacizumab(Avastin®), Ziv-aflibercept (Zaltrap®), Sorafenib (Nexavar®), Sunitinib(Sutent®), Pazopanib (Votrient®), Regorafenib (Stivarga®), andCabozantinib (Cometriq™).

In certain aspects, the additional therapeutic agent is ananti-inflammatory agent, a chemotherapeutic agent, a radiotherapeutic,additional therapeutic agents, or immunosuppressive agents.

Suitable chemotherapeutic agents include, but are not limited to,radioactive molecules, toxins, also referred to as cytotoxins orcytotoxic agents, which includes any agent that is detrimental to theviability of cells, agents, and liposomes or other vesicles containingchemotherapeutic compounds. General anticancer pharmaceutical agentsinclude: Vincristine (Oncovin®) or liposomal vincristine (Marqibo®),Daunorubicin (daunomycin or Cerubidine®) or doxorubicin (Adriamycin®),Cytarabine (cytosine arabinoside, ara-C, or Cytosar®), L-asparaginase(Elspar®) or PEG-L-asparaginase (pegaspargase or Oncaspar®), Etoposide(VP-16), Teniposide (Vumon®), 6-mercaptopurine (6-MP or Purinethol®),Methotrexate, Cyclophosphamide (Cytoxan®), Prednisone, Dexamethasone(Decadron), imatinib (Gleevec®), dasatinib (Sprycel®), nilotinib(Tasigna®), bosutinib (Bosulif®), and ponatinib (Iclusig™) Examples ofadditional suitable chemotherapeutic agents include but are not limitedto 1-dehydrotestosterone, 5-fluorouracil decarbazine, 6-mercaptopurine,6-thioguanine, actinomycin D, adriamycin, aldesleukin, alkylatingagents, allopurinol sodium, altretamine, amifostine, anastrozole,anthramycin (AMC)), anti-mitotic agents, cis-dichlorodiamine platinum(II) (DDP) cisplatin), diamino dichloro platinum, anthracyclines,antibiotics, antimetabolites, asparaginase, BCG live (intravesical),betamethasone sodium phosphate and betamethasone acetate, bicalutamide,bleomycin sulfate, busulfan, calcium leucouorin, calicheamicin,capecitabine, carboplatin, lomustine (CCNU), carmustine (BSNU),Chlorambucil, Cisplatin, Cladribine, Colchicin, conjugated estrogens,Cyclophosphamide, Cyclothosphamide, Cytarabine, Cytarabine, cytochalasinB, Cytoxan, Dacarbazine, Dactinomycin, dactinomycin (formerlyactinomycin), daunirubicin HCL, daunorucbicin citrate, denileukindiftitox, Dexrazoxane, Dibromomannitol, dihydroxy anthracin dione,Docetaxel, dolasetron mesylate, doxorubicin HCL, dronabinol, E. coliL-asparaginase, emetine, epoetin-α, Erwinia L-asparaginase, esterifiedestrogens, estradiol, estramustine phosphate sodium, ethidium bromide,ethinyl estradiol, etidronate, etoposide citrororum factor, etoposidephosphate, filgrastim, floxuridine, fluconazole, fludarabine phosphate,fluorouracil, flutamide, folinic acid, gemcitabine HCL, glucocorticoids,goserelin acetate, gramicidin D, granisetron HCL, hydroxyurea,idarubicin HCL, ifosfamide, interferon α-2b, irinotecan HCL, letrozole,leucovorin calcium, leuprolide acetate, levamisole HCL, lidocaine,lomustine, maytansinoid, mechlorethamine HCL, medroxyprogesteroneacetate, megestrol acetate, melphalan HCL, mercaptipurine, mesna,methotrexate, methyltestosterone, mithramycin, mitomycin C, mitotane,mitoxantrone, nilutamide, octreotide acetate, ondansetron HCL,paclitaxel, pamidronate disodium, pentostatin, pilocarpine HCL,plimycin, polifeprosan 20 with carmustine implant, porfimer sodium,procaine, procarbazine HCL, propranolol, rituximab, sargramostim,streptozotocin, tamoxifen, taxol, teniposide, tenoposide, testolactone,tetracaine, thioepa chlorambucil, thioguanine, thiotepa, topotecan HCL,toremifene citrate, trastuzumab, tretinoin, valrubicin, vinblastinesulfate, vincristine sulfate, and vinorelbine tartrate.

Additional therapeutic agents that can be administered in combinationwith a compound disclosed herein can include bevacizumab, sutinib,sorafenib, 2-methoxyestradiol or 2ME2, finasunate, vatalanib,vandetanib, aflibercept, volociximab, etaracizumab (MEDI-522),cilengitide, erlotinib, cetuximab, panitumumab, gefitinib, trastuzumab,dovitinib, figitumumab, atacicept, rituximab, alemtuzumab, aldesleukine,atlizumab, tocilizumab, temsirolimus, everolimus, lucatumumab,dacetuzumab, HLL1, huN901-DM1, atiprimod, natalizumab, bortezomib,carfilzomib, marizomib, tanespimycin, saquinavir mesylate, ritonavir,nelfinavir mesylate, indinavir sulfate, belinostat, panobinostat,mapatumumab, lexatumumab, dulanermin, ABT-737, oblimersen, plitidepsin,talmapimod, P276-00, enzastaurin, tipifarnib, perifosine, imatinib,dasatinib, lenalidomide, thalidomide, simvastatin, and celecoxib.

In one aspect of the present invention, a compound described herein canbe combined with at least one immunosuppressive agent. Theimmunosuppressive agent is preferably selected from the group consistingof a calcineurin inhibitor, e.g. a cyclosporin or an ascomycin, e.g.Cyclosporin A (NEORAL®), FK506 (tacrolimus), pimecrolimus, a mTORinhibitor, e.g. rapamycin or a derivative thereof, e.g. Sirolimus(RAPAMUNE®), Everolimus (Certican®), temsirolimus, zotarolimus,biolimus-7, biolimus-9, a rapalog, e.g. ridaforolimus, azathioprine,campath 1H, a S1P receptor modulator, e.g. fingolimod or an analoguethereof, an anti IL-8 antibody, mycophenolic acid or a salt thereof,e.g. sodium salt, or a prodrug thereof, e.g. Mycophenolate Mofetil(CELLCEPT®), OKT3 (ORTHOCLONE OKT3®), Prednisone, ATGAM®,THYMOGLOBULIN®, Brequinar Sodium, OKT4, T10B9.A-3A, 33B3.1,15-deoxyspergualin, tresperimus, Leflunomide ARAVA®, CTLAI-Ig,anti-CD25, anti-IL2R, Basiliximab (SIMULECT®), Daclizumab (ZENAPAX®),mizorbine, methotrexate, dexamethasone, ISAtx-247, SDZ ASM 981(pimecrolimus, Elidel®), CTLA4lg (Abatacept), belatacept, LFA3lg,etanercept (sold as Enbrel® by Immunex), adalimumab (Humira®),infliximab (Remicade®), an anti-LFA-1 antibody, natalizumab (Antegren®),Enlimomab, gavilimomab, antithymocyte immunoglobulin, siplizumab,Alefacept efalizumab, pentasa, mesalazine, asacol, codeine phosphate,benorylate, fenbufen, naprosyn, diclofenac, etodolac and indomethacin,aspirin and ibuprofen.

In certain embodiments, a compound described herein is administered tothe subject prior to treatment with another chemotherapeutic agent,during treatment with another chemotherapeutic agent, afteradministration of another chemotherapeutic agent, or a combinationthereof.

In some embodiments, the selective compound can be administered to thesubject such that the other chemotherapeutic agent can be administeredeither at higher doses (increased chemotherapeutic dose intensity) ormore frequently (increased chemotherapeutic dose density). Dose-densechemotherapy is a chemotherapy treatment plan in which drugs are givenwith less time between treatments than in a standard chemotherapytreatment plan. Chemotherapy dose intensity represents unit dose ofchemotherapy administered per unit time. Dose intensity can be increasedor decreased through altering dose administered, time interval ofadministration, or both.

In one embodiment of the invention, the compounds described herein canbe administered in a concerted regimen with another agent such as anon-DNA-damaging, targeted anti-neoplastic agent or a hematopoieticgrowth factor agent. It has been recently been reported that theuntimely administration of hematopoietic growth factors can have seriousside effects. For example, the use of the EPO family of growth factorshas been associated with arterial hypertension, cerebral convulsions,hypertensive encephalopathy, thromboembolism, iron deficiency, influenzalike syndromes and venous thrombosis. The G-CSF family of growth factorshas been associated with spleen enlargement and rupture, respiratorydistress syndrome, allergic reactions and sickle cell complications. Bycombining the administration of the short-lived selective compoundsdescribed herein and methods of the present invention with the timelyadministration of hematopoietic growth factors, for example, at the timepoint wherein the affected cells are no longer under growth arrest, itis possible for the health care practitioner to decrease the amount ofthe growth factor to minimize the unwanted adverse effects whileachieving the desired therapeutic benefit. In one embodiment, the growthfactor is administered upon cessation of the effect of the compound onthe CDK4/6 replication dependent healthy cells, for example HSPCs. Thus,in this embodiment, the use of a selective compound described herein inan anti-neoplastic therapeutic regime may allow the subject to receive areduced amount of growth factor because the targeted hematopoietic cellswill have reentered the cell cycle quicker than when other CDK4/6inhibitors, for example PD0332991. In addition, rapid cell-cycle reentryfollowing G1 arrest using a compound described herein provides for theability to time the administration of hematopoietic growth factors toassist in the reconstitution of hematopoietic cell lines to maximize thegrowth factor effect, that is, when the growth factors will be mosteffective. As such, in one embodiment, the use of the compounds ormethods described herein is combined with the use of hematopoieticgrowth factors including, but not limited to, granulocyte colonystimulating factor (G-CSF, for example, sold as Neupogen (filgrastin),Neulasta (peg-filgrastin), or lenograstin), granulocyte-macrophagecolony stimulating factor (GM-CSF, for example sold as molgramostim andsargramostim (Leukine)), M-CSF (macrophage colony stimulating factor),thrombopoietin (megakaryocyte growth development factor (MGDF), forexample sold as Romiplostim and Eltrombopag) interleukin (IL)-12,interleukin-3, interleukin-11 (adipogenesis inhibiting factor oroprelvekin), SCF (stem cell factor, steel factor, kit-ligand, or KL) anderythropoietin (EPO), and their derivatives (sold as for exampleepoetin-α as Darbopoetin, Epocept, Nanokine, Epofit, Epogin, Eprex andProcrit; epoetin-β sold as for example NeoRecormon, Recormon andMicera), epoetin-delta (sold as for example Dynepo), epoetin-omega (soldas for example Epomax), epoetin zeta (sold as for example Silapo andReacrit) as well as for example Epocept, EPOTrust, Erypro Safe,Repoeitin, Vintor, Epofit, Erykine, Wepox, Espogen, Relipoeitin,Shanpoietin, Zyrop and EPIAO). In one embodiment, the CDK4/6 inhibitoris administered prior to administration of the hematopoietic growthfactor. In one embodiment, the hematopoietic growth factoradministration is timed so that the compound's effect on HSPCs hasdissipated. In one embodiment, the growth factor is administered atleast 20 hours after the administration of a compound described herein.

If desired, multiple doses of a compound described herein can beadministered to the subject. Alternatively, the subject can be given asingle dose of a compound described herein. For example, a compound canbe administered so that CDK4/6-replication dependent healthy cells areG1 arrested wherein, due to the rapid dissipation of the G1-arrestingeffect of the compounds, a significant number of healthy cells reenterthe cell-cycle and are capable of replicating shortly after exposure,for example, within about 24-48 hours or less, and continue to replicateuntil a following administration of the compound. In one embodiment, thecompound is administered to allow for the cycling of theCDK4/6-replication dependent healthy cells between G1-arrest and reentryinto the cell-cycle to accommodate a repeated-dosing treatment regimen,for example a long term repeated-dosing treatment regime.

In some embodiments, the CDK4/6-replication dependent healthy cells canbe arrested for longer periods, for example, over a period of hours,days, weeks and/or months, through multiple, limited-time separatedadministrations of a compound described herein. Because of the rapidreentry into the cell cycle by CDK4/6-replication dependent healthycells, for example HSPCs, upon dissipation of the compounds inhibitoryintra-cellular effects, the cells are capable of reconstituting the celllineages faster than CDK4/6 inhibitors with longer G1 arrestingprofiles, for example PD0332991.

The reduction in side effects, in particular myelosuppression, affordedby the compounds described herein can allow for dose intensification(e.g., more therapy can be given in a fixed period of time), which willtranslate to better efficacy. Therefore, the presently disclosed methodscan result in regimens that are less toxic and more effective. Whenappropriate, the small molecules can be formulated for oral, topical,intranasal, inhalation, intravenous or any other desired form ofadministration.

A compound useful in the methods described herein is a selective CDK4/6inhibitor compound that selectively inhibit at least one of CDK4 andCDK6 or through the inhibition of cellular replication of an Rb-positivecancer. In one embodiment, the compounds described herein have an IC₅₀for CDK4 as measured in a CDK4/CycD1 IC₅₀ phosphorylation assay that isat least 1500 times or greater lower than the compound's IC₅₀s for CDK2as measured in a CDK2/CycE IC₅₀ phosphorylation assay. In oneembodiment, the CDK4/6 inhibitors are at least about 10 times or greatermore potent (i.e., have an IC₅₀ in a CDK4/CycD1 phosphorylation assaythat is at least 10 times or more lower) than PD0332991.

The use of a compound as described herein can induce selective G1 arrestin CDK4/6-dependent cells (e.g., as measured in a cell-based in vitroassay). In one embodiment, the CDK4/6 inhibitor is capable of increasingthe percentage of CDK4/6-dependent cells in the G1 phase, whiledecreasing the percentage of CDK4/6-dependent cells in the G2/M phaseand S phase. In one embodiment, the compound induces substantially pure(i.e., “clean”) G1 cell cycle arrest in the CDK4/6-dependent cells,e.g., wherein treatment with the compound induces cell cycle arrest suchthat the majority of cells are arrested in G1 as defined by standardmethods (e.g. propidium iodide (PI) staining or others) with thepopulation of cells in the G2/M and S phases combined being less thanabout 30%, about 25%, about 20%, about 15%, about 10%, about 5%, about3% or less of the total cell population. Methods of assessing the cellphase of a population of cells are known in the art (see, for example,in U.S. Patent Application Publication No. 2002/0224522) and includecytometric analysis, microscopic analysis, gradient centrifugation,elutriation, fluorescence techniques including immunofluorescence, andcombinations thereof. Cytometric techniques include exposing the cell toa labeling agent or stain, such as DNA-binding dyes, e.g., PI, andanalyzing cellular DNA content by flow cytometry. Immunofluorescencetechniques include detection of specific cell cycle indicators such as,for example, thymidine analogs (e.g., 5-bromo-2-deoxyuridine (BrdU) oran iododeoxyuridine), with fluorescent antibodies.

In some embodiments, the use of a compound described herein result inreduced or substantially free of off-target effects, particularlyrelated to inhibition of kinases other than CDK4 and or CDK6 such asCDK2, as the compounds described herein are poor inhibitors (e.g., >1 uMIC₅₀) of CDK2. Furthermore, because of the high selectivity for CDK4/6,the use of the compounds described herein should not induce cell cyclearrest in CDK4/6-independent cells. In addition, because of the shorttransient nature of the G1-arrest effect, the CDK4/6-replicationdependent healthy cells more quickly reenter the cell-cycle than,comparatively, use of PD0332991 provides, resulting in the reduced riskof, in one embodiment, hematological deficiency during long termtreatment regimens due to the ability of HSPCs to replicate betweentreatments.

In one aspect of the invention, a compound disclosed herein can bebeneficially administered in combination with any therapeutic regimenentailing radiotherapy, chemotherapy, or other therapeutic agents. Inadditional embodiments the compounds disclosed herein can bebeneficially administered in combination with therapeutic agentstargeting auto-immune disorders.

Drug Conjugates

In one embodiment, the activity of an active compound for a purposedescribed herein can be augmented through conjugation to an agent thattargets the diseased or abnormally proliferating cell or otherwiseenhances activity, delivery, pharmacokinetics or other beneficialproperty.

For example, the compound can be administered as an antibody-drugconjugates (ADC). In certain embodiments, a selected compound describedherein can be administered in conjugation or combination with anantibody or antibody fragment. Fragments of an antibody can be producedthrough chemical or genetic mechanisms. The antibody fragment can be anantigen binding fragment. For example, the antigen binding fragment canbe selected from an Fab, Fab′, (Fab′)2, or Fv. The antibody fragment canbe a Fab. Monovalent F(ab) fragments have one antigen binding site. Theantibody can be a divalent (Fab′)2 fragment, which has two antigenbinding regions that are linked by disulfide bonds. In one embodiment,the antigen fragment is a (Fab′). Reduction of F(ab′)2 fragmentsproduces two monovalent Fab′ fragments, which have a free sulfhydrylgroup that is useful for conjugation to other molecules.

A selected compound described herein can be administered in conjugationor combination with a Fv fragment. Fv fragments are the smallestfragment made from enzymatic cleavage of IgG and IgM class antibodies.Fv fragments have the antigen-binding site made of the VH and VCregions, but they lack the CH1 and CL regions. The VH and VL chains areheld together in Fv fragments by non-covalent interactions.

In one embodiment, a selected compound as described herein can beadministered in combination with an antibody fragment selected from thegroup consisting of an ScFv, domain antibody, diabody, triabody,tetrabody, Bis-scFv, minibody, Fab2, or Fab3 antibody fragment. In oneembodiment, the antibody fragment is a ScFv. Genetic engineering methodsallow the production of single chain variable fragments (ScFv), whichare Fv type fragments that include the VH and VL domains linked with aflexible peptide When the linker is at least 12 residues long, the ScFvfragments are primarily monomeric. Manipulation of the orientation ofthe V-domains and the linker length creates different forms of Fvmolecules Linkers that are 3-11 residues long yield scFv molecules thatare unable to fold into a functional Fv domain. These molecules canassociate with a second scFv molecule, to create a bivalent diabody. Inone embodiment, the antibody fragment administered in combination with aselected compound described herein is a bivalent diabody. If the linkerlength is less than three residues, scFv molecules associate intotriabodies or tetrabodies. In one embodiment, the antibody fragment is atriabody. In one embodiment, the antibody fragment is a tetrabody.Multivalent scFvs possess greater functional binding affinity to theirtarget antigens than their monovalent counterparts by having binding totwo more target antigens, which reduces the off-rate of the antibodyfragment. In one embodiment, the antibody fragment is a minibody.Minibodies are scFv-CH3 fusion proteins that assemble into bivalentdimers. In one embodiment, the antibody fragment is a Bis-scFv fragment.Bis-scFv fragments are bispecific. Miniaturized ScFv fragments can begenerated that have two different variable domains, allowing theseBis-scFv molecules to concurrently bind to two different epitopes.

In one embodiment, a selected compound described herein is administeredin conjugation or combination with a bispecific dimer (Fab2) ortrispecific dimer (Fab3). Genetic methods are also used to createbispecific Fab dimers (Fab2) and trispecific Fab trimers (Fab3). Theseantibody fragments are able to bind 2 (Fab2) or 3 (Fab3) differentantigens at once.

In one embodiment, a selected compound described herein is administeredin conjugation or combination with an rIgG antibody fragment. rIgGantibody fragments refers to reduced IgG (75,000 daltons) or half-IgG.It is the product of selectively reducing just the hinge-regiondisulfide bonds. Although several disulfide bonds occur in IgG, those inthe hinge-region are most accessible and easiest to reduce, especiallywith mild reducing agents like 2-mercaptoethylamine (2-MEA). Half-IgGare frequently prepared for the purpose of targeting the exposinghinge-region sulfhydryl groups that can be targeted for conjugation,either antibody immobilization or enzyme labeling.

In other embodiments, a selected active compound described herein can belinked to a radioisotope to increase efficacy, using methods well knownin the art. Any radioisotope that is useful against Rb-positive cancercells can be incorporated into the conjugate, for example, but notlimited to, ¹³¹I, ¹²³I, ¹⁹²Ir, ³²P, ⁹⁰Sr, ¹⁹⁸Au, ²²⁶Ra, ⁹⁰Y, ²⁴¹Am,²⁵²Cf, ⁶⁰Co, or ¹³⁷Cs.

Of note, the linker chemistry can be important to efficacy andtolerability of the drug conjugates. The thio-ether linked T-DM1increases the serum stability relative to a disulfide linker version andappears to undergo endosomal degradation, resulting in intra-cellularrelease of the cytotoxic agent, thereby improving efficacy andtolerability, See, Barginear, M. F. and Budman, D. R., Trastuzumab-DM1:A review of the novel immune-conjugate for HER2-overexpressing breastcancer, The Open Breast Cancer Journal, 1:25-30, 2009.

Examples of early and recent antibody-drug conjugates, discussing drugs,linker chemistries and classes of targets for product development thatmay be used in the present invention can be found in the reviews byCasi, G. and Neri, D., Antibody-drug conjugates: basic concepts,examples and future perspectives, J. Control Release 161(2):422-428,2012, Chari, R. V., Targeted cancer therapy: conferring specificity tocytotoxic drugs, Acc. Chem. Rev., 41(1):98-107, 2008, Sapra, P. andShor, B., Monoclonal antibody-based therapies in cancer: advances andchallenges, Pharmacol. Ther., 138(3):452-69, 2013, Schliemann, C. andNeri, D., Antibody-based targeting of the tumor vasculature, Biochim.Biophys. Acta., 1776(2):175-92, 2007, Sun, Y., Yu, F., and Sun, B. W.,Antibody-drug conjugates as targeted cancer therapeutics, Yao Xue XueBao, 44(9):943-52, 2009, Teicher, B. A., and Chari, R. V., Antibodyconjugate therapeutics: challenges and potential, Clin. Cancer Res.,17(20):6389-97, 2011, Firer, M. A., and Gellerman, G. J., Targeted drugdelivery for cancer therapy: the other side of antibodies, J. Hematol.Oncol., 5:70, 2012, Vlachakis, D. and Kossida, S., Antibody DrugConjugate bioinformatics: drug delivery through the letterbox, Comput.Math. Methods Med., 2013; 2013:282398, Epub 2013 Jun. 19, Lambert, J.M., Drug-conjugated antibodies for the treatment of cancer, Br. J. Clin.Pharmacol., 76(2):248-62, 2013, Concalves, A., Tredan, O., Villanueva,C. and Dumontet, C., Antibody-drug conjugates in oncology: from theconcept to trastuzumab emtansine (T-DM1), Bull. Cancer,99(12):1183-1191, 2012, Newland, A. M., Brentuximab vedotin: aCD-30-directed antibody-cytotoxic drug conjugate, Pharmacotherapy,33(1):93-104, 2013, Lopus, M., Antibody-DM1 conjugates as cancertherapeutics, Cancer Lett., 307(2):113-118, 2011, Chu, Y. W. and Poison,A., Antibody-drug conjugates for the treatment of B-cell non-Hodgkin'slymphoma and leukemia, Future Oncol., 9(3):355-368, 2013, Bertholjotti,I., Antibody-drug conjugate—a new age for personalized cancer treatment,Chimia, 65(9): 746-748, 2011, Vincent, K. J., and Zurini, M., Currentstrategies in antibody engineering: Fc engineering and pH—dependentantigen binding, bispecific antibodies and antibody drug conjugates,Biotechnol. J., 7(12):1444-1450, 2012, Haeuw, J. F., Caussanel, V., andBeck, A., Immunoconjugates, drug-armed antibodies to fight againstcancer, Med. Sci., 25(12):1046-1052, 2009 and Govindan, S. V., andGoldenberg, D. M., Designing immunoconjugates for cancer therapy, ExpertOpin. Biol. Ther., 12(7):873-890, 2012.

Pharmaceutical Compositions and Dosage Forms

An active compound described herein, or its salt, isotopic analog, orprodrug can be administered in an effective amount to the host using anysuitable approach which achieves the desired therapeutic result. Theamount and timing of active compound administered will, of course, bedependent on the host being treated, the instructions of the supervisingmedical specialist, on the time course of the exposure, on the manner ofadministration, on the pharmacokinetic properties of the particularactive compound, and on the judgment of the prescribing physician. Thus,because of host to host variability, the dosages given below are aguideline and the physician can titrate doses of the compound to achievethe treatment that the physician considers appropriate for the host. Inconsidering the degree of treatment desired, the physician can balance avariety of factors such as age and weight of the host, presence ofpreexisting disease, as well as presence of other diseases.Pharmaceutical formulations can be prepared for any desired route ofadministration including, but not limited to, oral, intravenous, oraerosol administration, as discussed in greater detail below.

The therapeutically effective dosage of any active compound describedherein will be determined by the health care practitioner depending onthe condition, size and age of the patient as well as the route ofdelivery. In one non-limited embodiment, a dosage from about 0.1 toabout 200 mg/kg has therapeutic efficacy, with all weights beingcalculated based upon the weight of the active compound, including thecases where a salt is employed. In some embodiments, the dosage can bethe amount of compound needed to provide a serum concentration of theactive compound of up to between about 1 and 5, 10, 20, 30, or 40 μM. Insome embodiments, a dosage from about 10 mg/kg to about 50 mg/kg can beemployed for oral administration. Typically, a dosage from about 0.5mg/kg to 5 mg/kg can be employed for intramuscular injection. In someembodiments, dosages can be from about 1 μmol/kg to about 50 μmol/kg,or, optionally, between about 22 μmol/kg and about 33 μmol/kg of thecompound for intravenous or oral administration. An oral dosage form caninclude any appropriate amount of active material, including for examplefrom 5 mg to, 50, 100, 200, or 500 mg per tablet or other solid dosageform.

In accordance with the presently disclosed methods, pharmaceuticallyactive compounds as described herein can be administered orally as asolid or as a liquid, or can be administered intramuscularly,intravenously, or by inhalation as a solution, suspension, or emulsion.In some embodiments, the compounds or salts also can be administered byinhalation, intravenously, or intramuscularly as a liposomal suspension.When administered through inhalation the active compound or salt can bein the form of a plurality of solid particles or droplets having anydesired particle size, and for example, from about 0.01, 0.1 or 0.5 toabout 5, 10, 20 or more microns, and optionally from about 1 to about 2microns. Compounds as disclosed in the present invention havedemonstrated good pharmacokinetic and pharmacodynamics properties, forinstance when administered by the oral or intravenous routes.

The pharmaceutical formulations can comprise an active compounddescribed herein or a pharmaceutically acceptable salt thereof, in anypharmaceutically acceptable carrier. If a solution is desired, water maybe the carrier of choice for water-soluble compounds or salts. Withrespect to the water-soluble compounds or salts, an organic vehicle,such as glycerol, propylene glycol, polyethylene glycol, or mixturesthereof, can be suitable. In the latter instance, the organic vehiclecan contain a substantial amount of water. The solution in eitherinstance can then be sterilized in a suitable manner known to those inthe art, and for illustration by filtration through a 0.22-micronfilter. Subsequent to sterilization, the solution can be dispensed intoappropriate receptacles, such as depyrogenated glass vials. Thedispensing is optionally done by an aseptic method. Sterilized closurescan then be placed on the vials and, if desired, the vial contents canbe lyophilized.

In addition to the active compounds or their salts, the pharmaceuticalformulations can contain other additives, such as pH-adjustingadditives. In particular, useful pH-adjusting agents include acids, suchas hydrochloric acid, bases or buffers, such as sodium lactate, sodiumacetate, sodium phosphate, sodium citrate, sodium borate, or sodiumgluconate. Further, the formulations can contain antimicrobialpreservatives. Useful antimicrobial preservatives include methylparaben,propylparaben, and benzyl alcohol. An antimicrobial preservative istypically employed when the formulations is placed in a vial designedfor multi-dose use. The pharmaceutical formulations described herein canbe lyophilized using techniques well known in the art.

For oral administration a pharmaceutical composition can take the formof solutions, suspensions, tablets, pills, capsules, powders, and thelike. Tablets containing various excipients such as sodium citrate,calcium carbonate and calcium phosphate may be employed along withvarious disintegrants such as starch (e.g., potato or tapioca starch)and certain complex silicates, together with binding agents such aspolyvinylpyrrolidone, sucrose, gelatin and acacia. Additionally,lubricating agents such as magnesium stearate, sodium lauryl sulfate,and talc are often very useful for tableting purposes. Solidcompositions of a similar type may be employed as fillers in soft andhard-filled gelatin capsules. Materials in this connection also includelactose or milk sugar as well as high molecular weight polyethyleneglycols. When aqueous suspensions and/or elixirs are desired for oraladministration, the compounds of the presently disclosed host matter canbe combined with various sweetening agents, flavoring agents, coloringagents, emulsifying agents and/or suspending agents, as well as suchdiluents as water, ethanol, propylene glycol, glycerin and various likecombinations thereof.

In yet another embodiment of the host matter described herein, there areprovided injectable, stable, sterile formulations comprising an activecompound as described herein, or a salt thereof, in a unit dosage formin a sealed container. The compound or salt is provided in the form of alyophilizate, which is capable of being reconstituted with a suitablepharmaceutically acceptable carrier to form liquid formulation suitablefor injection thereof into a host. When the compound or salt issubstantially water-insoluble, a sufficient amount of emulsifying agent,which is physiologically acceptable, can be employed in sufficientquantity to emulsify the compound or salt in an aqueous carrier.Particularly useful emulsifying agents include phosphatidyl cholines andlecithin.

Additional embodiments provided herein include liposomal formulations ofthe active compounds disclosed herein. The technology for formingliposomal suspensions is well known in the art. When the compound is anaqueous-soluble salt, using conventional liposome technology, the samecan be incorporated into lipid vesicles. In such an instance, due to thewater solubility of the active compound, the active compound can besubstantially entrained within the hydrophilic center or core of theliposomes. The lipid layer employed can be of any conventionalcomposition and can either contain cholesterol or can becholesterol-free. When the active compound of interest iswater-insoluble, again employing conventional liposome formationtechnology, the salt can be substantially entrained within thehydrophobic lipid bilayer that forms the structure of the liposome. Ineither instance, the liposomes that are produced can be reduced in size,as through the use of standard sonication and homogenization techniques.The liposomal formulations comprising the active compounds disclosedherein can be lyophilized to produce a lyophilizate, which can bereconstituted with a pharmaceutically acceptable carrier, such as water,to regenerate a liposomal suspension.

Pharmaceutical formulations also are provided which are suitable foradministration as an aerosol by inhalation. These formulations comprisea solution or suspension of a desired compound described herein or asalt thereof, or a plurality of solid particles of the compound or salt.The desired formulations can be placed in a small chamber and nebulized.Nebulization can be accomplished by compressed air or by ultrasonicenergy to form a plurality of liquid droplets or solid particlescomprising the compounds or salts. The liquid droplets or solidparticles may for example have a particle size in the range of about 0.5to about 10 microns, and optionally from about 0.5 to about 5 microns.In one embodiment, the solid particles provide for controlled releasethrough the use of a degradable polymer. The solid particles can beobtained by processing the solid compound or a salt thereof, in anyappropriate manner known in the art, such as by micronization.Optionally, the size of the solid particles or droplets can be fromabout 1 to about 2 microns. In this respect, commercial nebulizers areavailable to achieve this purpose. The compounds can be administered viaan aerosol suspension of respirable particles in a manner set forth inU.S. Pat. No. 5,628,984, the disclosure of which is incorporated hereinby reference in its entirety.

Pharmaceutical formulations also are provided which provide a controlledrelease of a compound described herein, including through the use of adegradable polymer, as known in the art.

When the pharmaceutical formulations suitable for administration as anaerosol is in the form of a liquid, the formulations can comprise awater-soluble active compound in a carrier that comprises water. Asurfactant can be present, which lowers the surface tension of theformulations sufficiently to result in the formation of droplets withinthe desired size range when hosted to nebulization.

The term “pharmaceutically acceptable salts” as used herein refers tothose salts which are, within the scope of sound medical judgment,suitable for use in contact with hosts (e.g., human hosts) without unduetoxicity, irritation, allergic response, and the like, commensurate witha reasonable benefit/risk ratio, and effective for their intended use,as well as the zwitterionic forms, where possible, of the compounds ofthe presently disclosed host matter.

Thus, the term “salts” refers to the relatively non-toxic, inorganic andorganic acid addition salts of the presently disclosed compounds. Thesesalts can be prepared during the final isolation and purification of thecompounds or by separately reacting the purified compound in its freebase form with a suitable organic or inorganic acid and isolating thesalt thus formed. Basic compounds are capable of forming a wide varietyof different salts with various inorganic and organic acids. Acidaddition salts of the basic compounds are prepared by contacting thefree base form with a sufficient amount of the desired acid to producethe salt in the conventional manner. The free base form can beregenerated by contacting the salt form with a base and isolating thefree base in the conventional manner. The free base forms may differfrom their respective salt forms in certain physical properties such assolubility in polar solvents. Pharmaceutically acceptable base additionsalts may be formed with metals or amines, such as alkali and alkalineearth metal hydroxides, or of organic amines. Examples of metals used ascations, include, but are not limited to, sodium, potassium, magnesium,calcium, and the like. Examples of suitable amines include, but are notlimited to, N,N′-dibenzylethylenediamine, chloroprocaine, choline,diethanolamine, ethylenediamine, N-methylglucamine, and procaine. Thebase addition salts of acidic compounds are prepared by contacting thefree acid form with a sufficient amount of the desired base to producethe salt in the conventional manner. The free acid form can beregenerated by contacting the salt form with an acid and isolating thefree acid in a conventional manner. The free acid forms may differ fromtheir respective salt forms somewhat in certain physical properties suchas solubility in polar solvents.

Salts can be prepared from inorganic acids sulfate, pyrosulfate,bisulfate, sulfite, bisulfite, nitrate, phosphate,monohydrogenphosphate, dihydrogenphosphate, metaphosphate,pyrophosphate, chloride, bromide, iodide such as hydrochloric, nitric,phosphoric, sulfuric, hydrobromic, hydriodic, phosphorus, and the like.Representative salts include the hydrobromide, hydrochloride, sulfate,bisulfate, nitrate, acetate, oxalate, valerate, oleate, palmitate,stearate, laurate, borate, benzoate, lactate, phosphate, tosylate,citrate, maleate, fumarate, succinate, tartrate, naphthylate mesylate,glucoheptonate, lactobionate, laurylsulphonate and isethionate salts,and the like. Salts can also be prepared from organic acids, such asaliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoicacids, hydroxy alkanoic acids, alkanedioic acids, aromatic acids,aliphatic and aromatic sulfonic acids, etc. and the like. Representativesalts include acetate, propionate, caprylate, isobutyrate, oxalate,malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate,benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate,benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate,maleate, tartrate, methanesulfonate, and the like. Pharmaceuticallyacceptable salts can include cations based on the alkali and alkalineearth metals, such as sodium, lithium, potassium, calcium, magnesium andthe like, as well as non-toxic ammonium, quaternary ammonium, and aminecations including, but not limited to, ammonium, tetramethylammonium,tetraethylammonium, methylamine, dimethylamine, trimethylamine,triethylamine, ethylamine, and the like. Also contemplated are the saltsof amino acids such as arginate, gluconate, galacturonate, and the like.See, for example, Berge et al., J. Pharm. Sci., 1977, 66, 1-19, which isincorporated herein by reference.

Preparation of Active Compounds Syntheses

The disclosed compounds can be made by the following general schemes:

In Scheme 1, Ref-1 is WO 2010/020675 A1; Ref-2 is White, J. D.; et al.J. Org. Chem. 1995, 60, 3600; and Ref-3 Presser, A. and Hufner, A.Monatshefte für Chemie 2004, 135, 1015.

In Scheme 2, Ref-1 is WO 2010/020675 A1; Ref-4 is WO 2005/040166 A1; andRef-5 is Schoenauer, K and Zbiral, E. Tetrahedron Letters 1983, 24, 573.

In Scheme 3, Ref—1 is WO 2010/020675 A1.

In Scheme 8, Ref-1 is WO 2010/020675 A1; Ref-2 is WO 2005/040166 A1; andRef-3 is Schoenauer, K and Zbiral, E. Tetrahedron Letters 1983, 24, 573.

Alternatively, the lactam can be generated by reacting the carboxylicacid with a protected amine in the presence of a strong acid and adehydrating agent, which can be together in one moiety as a strong acidanhydride. Examples of strong acid anhydrides include, but are notlimited to, trifluoroacetic acid anhydride, tribromoacetic acidanhydride, trichloroacetic acid anhydride, or mixed anhydrides. Thedehydrating agent can be a carbodiimide based compound such as but notlimited to DCC (N,N-dicyclohexylcarbodiimide), EDC(1-ethyl-3-(3-dimethylaminopropyl)carbodiimide or DIC(N,N-diisopropylcarbodiimide). An additional step may be necessary totake off the N-protecting group and the methodologies are known to thoseskilled in the art.

Alternatively, the halogen moiety bonded to the pyrimidine ring can besubstituted with any leaving group that can be displaced by a primaryamine, for example to create an intermediate for a final product such asBr, I, F, SMe, SO₂Me, SOalkyl, SO₂ alkyl. See, for ExamplePCT/US2013/037878 to Tavares.

Other amine intermediates and final amine compounds can be synthesizedby those skilled in the art. It will be appreciated that the chemistrycan employ reagents that comprise reactive functionalities that can beprotected and de-protected and will be known to those skilled in the artat the time of the invention. See for example, Greene, T. W. and Wuts,P. G. M., Greene's Protective Groups in Organic Synthesis, 4^(th)edition, John Wiley and Sons.

CDK4/6 Inhibitors of the present invention can be synthesized accordingto the generalized Scheme 9. Specific synthesis and characterization ofthe Substituted 2-aminopyrmidines can be found in, for instance,WO2012/061156.

Compounds T, Q, GG, and U were prepared as above and were characterizedby mass spectrometry and NMR as shown below:

Compound T

¹H NMR (600 MHz, DMSO-d₆) δ ppm 1.47 (br. s., 6H) 1.72 (br. s., 2H) 1.92(br. s., 2H) 2.77 (br. s., 3H) 3.18 (br. s., 2H) 3.46 (br. s., 2H) 3.63(br. s., 2H) 3.66 (d, J=6.15 Hz, 2H) 3.80 (br. s., 2H) 7.25 (s, 1H) 7.63(br. s., 2H) 7.94 (br. s., 1H) 8.10 (br. s., 1H) 8.39 (br. s., 1H) 9.08(br. s., 1H) 11.59 (br. s., 1H). LCMS ESI (M+H) 447.

Compound Q

¹H NMR (600 MHz, DMSO-d₆) δ ppm 0.82 (d, J=7.32 Hz, 2H) 1.08-1.37 (m,3H) 1.38-1.64 (m, 2H) 1.71 (br. s., 1H) 1.91 (br. s., 1H) 2.80 (br. s.,1H) 3.12 (s, 1H) 3.41 (br. s., 4H) 3.65 (br. s., 4H) 4.09 (br. s., 1H)7.26 (s, 1H) 7.52-7.74 (m, 2H) 7.94 (br. s., 1H) 8.13 (br. s., 1H) 8.40(br. s., 1H) 9.09 (br. s., 1H) 9.62 (br. s., 1H) 11.71 (br. s., 1H).LCMS ESI (M+H) 433.

Compound GG

¹H NMR (600 MHz, DMSO-d₆) δ ppm 0.85 (br. s., 1H) 1.17-1.39 (m, 7H)1.42-1.58 (m, 2H) 1.67-1.84 (m, 3H) 1.88-2.02 (m, 1H) 2.76-2.93 (m, 1H)3.07-3.22 (m, 1H) 3.29-3.39 (m, 1H) 3.41-3.61 (m, 4H) 3.62-3.76 (m, 4H)3.78-3.88 (m, 1H) 4.12 (br. s., 1H) 7.28 (s, 1H) 7.60-7.76 (m, 2H) 7.98(s, 1H) 8.13 (br. s., 1H) 8.41 (s, 1H) 9.10 (br. s., 1H) 11.21 (br. s.,1H) 11.54 (s, 1H). LCMS ESI (M+H) 475.

Compound U

¹H NMR (600 MHz, DMSO-d₆) δ ppm 0.84 (t, J=7.61 Hz, 2H) 1.13-1.39 (m,4H) 1.46 (d, J=14.05 Hz, 2H) 1.64-1.99 (m, 6H) 2.21 (br. s., 1H)2.66-2.89 (m, 2H) 3.06 (br. s., 1H) 3.24-3.36 (m, 1H) 3.37-3.50 (m, 2H)3.56-3.72 (m, 2H) 3.77-4.00 (m, 4H) 4.02-4.19 (m, 2H) 7.25 (s, 1H)7.50-7.75 (m, 2H) 7.89 (d, J=2.93 Hz, 1H) 8.14 (d, J=7.32 Hz, 1H) 8.38(br. s., 1 H) 9.06 (s, 1H) 11.53 (br. s., 1H). LCMS ESI (M+H) 517.

EXAMPLES

Intermediates B, E, K, L, 1A, 1F and 1CA were synthesized according toU.S. Pat. No. 8,598,186 entitled CDK Inhibitors to Tavares, F. X. andStrum, J. C.

The patents WO 2013/148748 entitled Lactam Kinase Inhibitors to Tavares,F. X., WO 2013/163239 entitled Synthesis of Lactams to Tavares, F. X.,and U.S. Pat. No. 8,598,186 entitled CDK Inhibitors to Tavares, F. X.and Strum, J. C. are incorporated by reference herein in their entirety.

Example 1 Synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4yl)amino]ethyl]carbamate, Compound 1

To a solution of 5-bromo-2,4-dichloropyrimidine (3.2 g, 0.0135 mol) inethanol (80 mL) was added Hunig's base (3.0 mL) followed by the additionof a solution of N-(tert-butoxycarbonyl)-1,2-diaminoethane (2.5 g,0.0156 mole) in ethanol (20 mL). The contents were stirred overnight for20 hrs. The solvent was evaporated under vacuum. Ethyl acetate (200 mL)and water (100 mL) were added and the layers separated. The organiclayer was dried with magnesium sulfate and then concentrated undervacuum. Column chromatography on silica gel using hexane/ethyl acetate(0-60%) afforded tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. ¹HNMR(d₆-DMSO) δ ppm 8.21 (s, 1H), 7.62 (brs, 1H), 7.27 (brs, 1H), 3.39 (m,2H), 3.12 (m, 2H), 1.34 (s, 9H). LCMS (ESI) 351 (M+H).

Example 2 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate,Compound 2

To tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate (1.265 g,3.6 mmol) in THF (10 mL) was added the acetal (0.778 mL, 5.43 mmol),Pd(dppf)CH₂Cl₂ (148 mg), and triethylamine (0.757 mL, 5.43 mmol). Thecontents were degassed and then purged with nitrogen. To this was thenadded CuI (29 mg). The reaction mixture was heated at reflux for 48 hrs.After cooling, the contents were filtered over CELITE™ and concentrated.Column chromatography of the resulting residue using hexane/ethylacetate (0-30%) afforded tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate.¹HNMR (d6-DMSO) δ ppm 8.18 (s, 1H), 7.63 (brs, 1H), 7.40 (brs, 1H), 5.55(s, 1H), 3.70 (m, 2H), 3.60 (m, 2H), 3.42 (m, 2H), 3.15 (m, 2H),1.19-1.16 (m, 15H). LCMS (ESI) 399 (M+H).

Example 3 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 3

To a solution of the coupled product (2.1 g, 0.00526 mole) in THF (30mL) was added TBAF solid (7.0 g). The contents were heated to andmaintained at 65 degrees for 2 hrs. Concentration followed by columnchromatography using ethyl acetate/hexane (0-50%) afforded tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamateas a pale brown liquid (1.1 g). ¹HNMR (d6-DMSO) δ ppm 8.88 (s, 1H), 6.95(brs, 1H), 6.69 (s, 1H), 5.79 (s, 1H), 4.29 (m, 2H), 3.59 (m, 4H), 3.34(m, 1H), 3.18 (m, 1H), 1.19 (m, 9H), 1.17 (m, 6H). LCMS (ESI) 399 (M+H).

Example 4 Synthesis of tert-butylN-[2-(2-chloro-6-formyl-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]carbamate,Compound 4

To the acetal (900 mg) from the preceeding step was added AcOH (8.0 mL)and water (1.0 mL). The reaction was stirred at room temperature for 16hrs. Conc. and column chromatography over silica gel using ethylacetate/hexanes (0-60%) afforded tert-butylN-[2-(2-chloro-6-formyl-pyrrolo[2,3-d]pyrimidin-7-yl)ethyl]carbamate asa foam (0.510 g). ¹HNMR (d6-DMSO) δ ppm 9.98 (s, 1H), 9.18 (s, 1H), 7.66(s, 1H), 6.80 (brs, 1H), 4.52 (m, 2H), 4.36 (m, 2H), 1.14 (s, 9H). LCMS(ESI) 325 (M+H).

Example 5 Synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 5

To the aldehyde (0.940 g) from the preceeding step in DMF (4 mL) wasadded oxone (1.95 g, 1.1 eq). The contents were stirred at room temp for7 hrs. Silica gel column chromatography using hexane/ethyl acetate(0-100%) afforded7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.545 g). ¹HNMR (d6-DMSO) δ ppm 9.11 (s, 1H), 7.39 (s, 1H), 4.38(m, 2H), 4.15 (m, 2H), 1.48 (m, 9H). LCMS (ESI) 341 (M+H).

Example 6 Synthesis of methyl7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylate,Compound 6

To a solution of 2-chloro-7-propyl-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.545 g, 0.00156 mole) from the preceeding step in toluene (3.5mL) and MeOH (1 mL) was added TMS-diazomethane (1.2 mL). After stirringovernight at room temperature, the excess of TMS-diazomethane wasquenched with acetic acid (3 mL) and the reaction was concentrated undervacuum. The residue was purified by silica gel column chromatographywith hexane/ethyl acetate (0-70%) to afford methyl7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylateas an off white solid (0.52 g). ¹HNMR (d6-DMSO) δ ppm 9.10 (s, 1H), 7.45(s, 1H), 6.81 (brs, 1H) 4.60 (m, 2H), 3.91 (s, 3H), 3.29 (m, 2H), 1.18(m, 9H) LCMS (ESI) 355 (M+H).

Example 7 Synthesis of Chloro Tricyclic Amide, Compound 7

To methyl7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylate(0.50 g, 0.0014 mole) from the preceeding step in dichloromethane (2.0mL) was added TFA (0.830 mL). The contents were stirred at roomtemperature for 1 hr. Concentration under vacuum afforded the crudeamino ester which was suspended in toluene (5 mL) and Hunig's base (0.5mL). The contents were heated at reflux for 2 hrs. Concentrationfollowed by silica gel column chromatography using hexane/ethyl acetate(0-50%) afforded the desired chloro tricyclic amide (0.260 g). ¹HNMR(d6-DMSO) δ ppm 9.08 (s, 1H), 8.48 (brs, 1H), 7.21 (s, 1H) 4.33 (m, 2H),3.64 (m, 2H). LCMS (ESI) 223 (M+H).

Example 8 Synthesis of Chloro-N-Methyltricyclic Amide, Compound 8

To a solution of the chloro tricycliclactam, Compound 7, (185 mg,0.00083 mole) in DMF (2.0 mL) was added sodium hydride (55% dispersionin oil, 52 mg). After stirring for 15 mins, methyl iodide (62 μL, 1.2eq). The contents were stirred at room temperature for 30 mins. Afterthe addition of methanol (5 mL), sat NaHCO₃ was added followed by theaddition of ethyl acetate. Separation of the organic layer followed bydrying with magnesium sulfate and concentration under vacuum affordedthe N-methylated amide in quantitative yield. ¹HNMR (d6-DMSO) δ ppm 9.05(s, 1H), 7.17 (s, 1H) 4.38 (m, 2H), 3.80 (m, 2H), 3.05 (s, 3H). LCMS(ESI) 237 (M+H).

Example 9 Synthesis of 1-Methyl-4-(6-Nitro-3-Pyridyl)Piperazine,Compound 9

To 5-bromo-2-nitropyridine (4.93 g, 24.3 mmole) in DMF (20 mL) was addedN-methylpiperazine (2.96 g, 1.1 eq) followed by the addition of DIPEA(4.65 mL, 26.7 mmole). The contents were heated at 90 degrees for 24hrs. After addition of ethyl acetate (200 mL), water (100 mL) was addedand the layers separated. Drying followed by concentration afforded thecrude product which was purified by silica gel column chromatographyusing (0-10%) DCM/Methanol. ¹HNMR (d6-DMSO) δ ppm 8.26 (s, 1H), 8.15(1H, d, J=9.3 Hz), 7.49 (1H, d, J=9.4 Hz), 3.50 (m, 4H), 2.49 (m, 4H),2.22 (s, 3H).

Example 10 Synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine,Compound 10

To 1-methyl-4-(6-nitro-3-pyridyl)piperazine (3.4 g) in ethyl acetate(100 mL) and ethanol (100 mL) was added 10% Pd/C (400 mg) and then thereaction was stirred under hydrogen (10 psi) overnight. After filtrationthrough CELITE™, the solvents were evaporated and the crude product waspurified by silica gel column chromatography using DCM/7N ammonia inMeOH (0-5%) to afford 5-(4-methylpiperazin-1-yl)pyridin-2-amine (2.2 g).¹HNMR (d6-DMSO) δ ppm 7.56 (1H, d, J=3 Hz), 7.13 (1H, m), 6.36 (1H, d,J=8.8 Hz), 5.33 (brs, 2H), 2.88 (m, 4H), 2.47 (m, 4H), 2.16 (s, 3H).

Example 11 Synthesis of tert-butyl4-(6-amino-3-pyridyl)piperazine-1-carboxylate, Compound 11

This compound was prepared as described in WO 2010/020675 A1.

Example 12 Synthesis of tert-butylN-[2-(benzyloxycarbonylamino)-3-methyl-butyl] carbamate, Compound 12

To benzyl N-[1-(hydroxymethyl)-2-methyl-propyl]carbamate (11.0 g, 0.0464mole) in dioxane (100 mL) cooled to 0° C. was added diphenylphosphorylazide (10.99 mL, 1.1 eq) followed by the addition of DBU (8.32 mL, 1.2eq). The contents were allowed to warm to room temperature and stirredfor 16 hrs. After the addition of ethyl acetate (300 mL) and water (100mL), the organic layer was separated and washed with satd. NaHCO₃ (100mL). The organic layer was then dried (magnesium sulfate) andconcentrated under vacuum. To this intermediate in DMSO (100 mL) wasadded sodium azide (7.54 g) and the contents then heated to 90 degreesfor 2 hrs. After addition of ethyl acetate and water the layers wereseparated. The organic layer was dried with magnesium sulfate followedby concentration under vacuum to afford an oil that was purified bysilica gel column chromatography using hexane/ethyl acetate (0-70%) toafford benzyl N-[1-(azidomethyl)-2-methyl-propyl] carbamate 6.9 g as acolorless oil.

To benzyl N-[1-(azidomethyl)-2-methyl-propyl] carbamate (6.9 g, 0.0263mole) in THF (100 mL) was added triphenyl phosphine (7.59 g, 1.1 eq).The contents were stirred for 20 hrs. After addition of water (10 mL),and stirring for an additional 6 hrs, ethyl acetate was added and thelayers separated. After drying with magnesium sulfate and concentrationunder vacuum, the crude product was purified by silica gel columnchromatography using DCM/MeOH (0-10%) to afford benzylN-[1-(aminomethyl)-2-methyl-propyl] carbamate as a yellow oil.

To benzyl N-[1-(aminomethyl)-2-methyl-propyl] carbamate (4.65 g, 0.019mole) in THF (70 mL) was added 2N NaOH (20 mL) followed by the additionof di-tert-butyl dicarbonate (5.15 g, 1.2 eq). After stirring for 16hrs, ethyl acetate was added and the layers separated. After drying withmagnesium sulfate and concentration under vacuum, the crude product waspurified using hexane/ethyl acetate (0-40%) over a silica gel column toafford intermediate A, tert-butylN-[2-(benzyloxycarbonylamino)-3-methyl-butyl] carbamate, (6.1 g). ¹HNMR(600 MHz, CHLOROFORM-d) δ ppm 0.89 (d, J=6.73 Hz, 3H) 0.92 (d, J=6.73Hz, 3H) 1.38 (s, 9H) 1.70-1.81 (m, 1H) 3.18 (d, J=5.56 Hz, 2H) 3.47-3.60(m, 1H) 4.76 (s, 1H) 4.89 (d, J=7.90 Hz, 1H) 5.07 (s, 2H) 7.25-7.36 (m,5H). LCMS (ESI) 337 (M+H).

Example 13 Synthesis of tert-butylN-[2-(benzyloxycarbonylamino)-4-methyl-pentyl] carbamate, Compound 13

To a solution of benzyl N-[1-(hydroxymethyl)-3-methyl-butyl]carbamate(6.3 g, 0.025 mole) in DCM (100 mL) was added diisopropylethyl amine(5.25 mL, 1.2 eq) followed by the addition of methane sulfonylchloride(2.13 mL, 1.1 eq) at 0 degrees. After stirring for 3 hrs, water (100 mL)was added and the organic layer separated. After drying with magnesiumsulfate and concentration under vacuum, the crude[2-(benzyloxycarbonylamino)-4-methyl-pentyl] methanesulfonate which wastaken directly to the next step.

To the crude [2-(benzyloxycarbonylamino)-4-methyl-pentyl]methanesulfonate from the above reaction in DMF (50 mL), was addedsodium azide 2.43 g. The reaction mixture was then heated to 85 degreesfor 3 hrs. After cooling, ethyl acetate (300 mL) and water was added.The organic layer was separated, dried with magnesium sulfate and thenconcentrated under vacuum to afford the crude benzylN-[1-(azidomethyl)-3-methyl-butyl] carbamate. To this crude intermediatewas added THF (100 mL) followed by triphenylphosphine 7.21 g and stirredunder nitrogen for 16 hrs. After addition of water (10 mL), and stirringfor an additional 6 hrs, ethyl acetate was added and the layersseparated. After drying with magnesium sulfate and concentration undervacuum, the crude product was columned using DCM/MeOH (0-10%) to affordbenzyl N-[1-(aminomethyl)-3-methyl-butyl] carbamate (4.5 g).

To benzyl N-[1-(aminomethyl)-3-methyl-butyl] carbamate (4.5 g, 0.018mole) in THF (60 mL) was added 2N NaOH (18 mL) followed by the additionof di-tert-butyl dicarbonate (4.19 g, 1.07 eq). After stirring for 16hrs, ethyl acetate was added and the layers separated. After drying withmagnesium sulfate and concentration under vacuum, the crude product wastaken to the next step. ¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.89 (d,J=6.73 Hz, 6H) 1.25-1.34 (m, 1H) 1.39 (s, 9H) 1.57-1.71 (m, 2H)3.04-3.26 (m, 2H) 3.68-3.80 (m, 1H) 4.72-4.89 (m, 2H) 5.06 (s, 2H)7.25-7.38 (m, 5H). LCMS (ESI) 351 (M+H).

Example 14 Synthesis of tert-butylN-[(2R)-2-(benzyloxycarbonylamino)-3-methyl-butyl] carbamate, Compound14

Compound 14 was synthesized from benzylN-[(1R)-1-(hydroxymethyl)-2-methyl-propyl] carbamate using similarsynthetic steps as that described for Compound 13. The analytical data(NMR and mass spec) was consistent with that for Compound 12.

Example 15 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-3-methyl-butyl]carbamate, Compound 15

Compound 15 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-2-methyl-propyl] carbamate using similarsynthetic steps as that described for Compound 13. The analytical data(NMR and mass spec) was consistent with that for Compound 12.

Example 16 Synthesis of tert-butylN-[(1S)-1-(aminomethyl)-2-methyl-propyl]carbamate, Compound 16

To a solution of tert-butylN-[(1S)-1-(hydroxymethyl)-2-methyl-propyl]carbamate carbamate (6.3 g,0.025 mole) in THF (100 mL) was added diisopropylethyl amine (5.25 mL,1.2 eq) followed by the addition of methane sulfonylchloride (2.13 mL,1.1 eq) at 0 degrees. After stirring for 3 hrs, water (100 mL) was addedand the organic layer separated. After drying with magnesium sulfate andconcentration under vacuum, the crude[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl] methanesulfonate wastaken directly to the next step.

To the crude [(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl]methanesulfonate from the above reaction in DMSO (50 mL), was addedsodium azide (2.43 g). The reaction mixture was then heated to 85degrees for 3 hrs. After cooling, ethyl acetate (300 mL) and water wereadded. The organic layer was separated, dried with magnesium sulfate andthen concentrated under vacuum to afford the crude benzylN-[1-(azidomethyl)-3-methyl-butyl] carbamate. To this crude intermediatewas added THF (100 mL) followed by triphenylphosphine (7.21 g) and thereaction was stirred under nitrogen for 16 hrs. After addition of water(10 mL), and stirring for an additional 6 hrs, ethyl acetate was addedand the layers separated. After drying with magnesium sulfate andconcentration under vacuum, the crude product was purified by silica gelcolumn chromatography using DCM/MeOH (0-10%) to afford benzylN-[1-(aminomethyl)-3-methyl-butyl] carbamate (4.5 g). LCMS (ESI) 203(M+H).

Example 17 Synthesis of tert-butylN-[(1R)-1-(aminomethyl)-2-methyl-propyl]carbamate, Compound 17

Compound 17 was synthesized from tert-butylN-[(1R)-1-(hydroxymethyl)-2-methyl-propyl] carbamate using a similarsynthetic sequence as described for Compound 16. The analytical data(NMR and mass spec) was consistent with Compound 16.

Example 18 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-4-methyl-pentyl] carbamate, Compound18

Compound 18 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-3-methyl-butyl]carbamate using a similarsynthetic sequence as described for Compound 13. The analytical data(NMR and mass spec) was consistent with Compound 13.

Example 19 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-2-phenyl-ethyl] carbamate, Compound19

Compound 19 was synthesized from benzylN-[(1S)-2-hydroxy-1-phenyl-ethyl] carbamate using a similar syntheticsequence as described for Compound 13. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.20-1.33 (m, 9H) 3.11 (t, J=6.29 Hz, 2H) 4.59-4.68 (m, 1H) 4.88-5.01(m, 2H) 6.81 (t, J=5.42 Hz, 1H) 7.14-7.35 (m, 10H) 7.69 (d, J=8.49 Hz,1H). LCMS (ESI) 371 (M+H).

Example 20 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-3-methyl-pentyl] carbamate, Compound20

Compound 20 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-2-methyl-butyl] carbamate using a similarsynthetic sequence as described for Compound 13. ¹HNMR (600 MHz,CHLOROFORM-d) δ ppm 0.85-0.92 (m, 6H) 1.05-1.15 (m, 1H) 1.35-1.41 (m,9H) 1.45-1.56 (m, 2H) 3.14-3.24 (m, 2H) 3.54-3.64 (m, 1H) 4.78 (s, 1H)4.96 (d, J=7.91 Hz, 1H) 5.06 (s, 2H) 7.27-7.37 (m, 5H). LCMS (ESI) 351(M+H).

Example 21 Synthesis of tert-butylN-[(2S)-2-(benzyloxycarbonylamino)-3,3-dimethyl-butyl] carbamate,Compound 21

Compound 21 was synthesized from benzylN-[(1S)-1-(hydroxymethyl)-2,2-dimethyl-propyl]carbamate using a similarsynthetic sequence as described for Compound 13. LCMS (ESI) 351.

Example 22 Synthesis of tert-butylN-[[1-(benzyloxycarbonylamino)cyclohexyl]methyl]carbamate, Compound 22

To a solution of benzyl N-[1-(aminomethyl)cyclohexyl]carbamate (10.0 g,0.0381 mole) in THF (150 mL) was added di-tert-butyl dicarbonate (9.15g, 1.1 eq) and the contents were stirred at room temperature for 16 hrs.Ethyl acetate and water were then added. The organic layer wasseparated, dried over magnesium sulfate and then concentrated undervacuum to afford tert-butylN-[[1-(benzyloxycarbonylamino)cyclohexyl]methyl] carbamate (13.1 g).¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.92-1.54 (m, 17H) 1.76-2.06 (m, 2H) 3.09(d, J=6.15 Hz, 2H) 4.92 (s, 2H) 6.63 (d, J=17.27 Hz, 1H) 7.16-7.49 (m,6H). LCMS (ESI) 363 (M+H).

Example 23 Synthesis of tert-butylN-[[1-(benzyloxycarbonylamino)cyclopentyl]methyl] carbamate, Compound 23

tert-butyl N-[[1-(benzyloxycarbonylamino)cyclopentyl]methyl]carbamatewas synthesized in an analogous manner to tert-butylN-[[1-(benzyloxycarbonylamino) cyclohexyl]methyl] carbamate. LCMS (ESI)349 (M+H).

Example 24 Synthesis of 2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine,Compound 24

To 5-bromo-2-nitropyridine (1.2 g, 5.9 mmol) in DMSO (4 mL) was added1-(4-piperidyl)piperidine (1.0 g, 5.9 mmole) and triethylamine (0.99 mL,7.1 mmole). The contents were heated to 120° C. in a CEM Discoverymicrowave system for 3 hours. The crude reaction was then purified bysilica gel column chromatography with DCM/methanol (0-20%) to afford2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine as an oil (457 mg).¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.26-1.36 (m, 2H) 1.43 (m, 6H) 1.76 (m,2H) 2.37 (m, 5H) 2.94 (t, J=12.74 Hz, 2H) 4.06 (d, J=13.47 Hz, 2H) 7.41(dd, J=9.37, 2.64 Hz, 1H) 8.08 (d, J=9.37 Hz, 1H) 8.20 (d, J=2.64 Hz,1H).

Example 25 Synthesis of 5-[4-(1-piperidyl)-1-piperidyl]pyridin-2-amine,Compound 25

5-[4-(1-piperidyl)-1-piperidyl]pyridin-2-amine was prepared in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.13-1.37 (m, 6H) 1.40-1.63 (m, 6H) 1.71 (m, 2H), 2.24 (m, 1H) 2.43(m, 2H) 3.33 (d, J=12.30 Hz, 2H) 5.31 (s, 2H) 6.33 (d, J=8.78 Hz, 1H)7.10 (dd, J=8.78, 2.93 Hz, 1H) 7.55 (d, J=2.64 Hz, 1H). LCMS (ESI) 261(M+H).

Example 26 Synthesis of 4-[1-(6-nitro-3-pyridyl)-4-piperidyl]morpholine, Compound 26

4-[1-(6-nitro-3-pyridyl)-4-piperidyl]morpholine was synthesized in amanner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.41 (m, 2H) 1.82 (m, 2H) 2.42 (m, 5H) 2.98 (t, J=12.44Hz, 2H) 3.52 (s, 4H) 4.04 (d, J=12.88 Hz, 2H) 7.42 (d, J=9.37 Hz, 1H)8.08 (d, J=9.08 Hz, 1H) 8.21 (s, 1H).

Example 27 Synthesis of 5-(4-morpholino-1-piperidyl) pyridin-2-amine,Compound 27

5-(4-morpholino-1-piperidyl)pyridin-2-amine was prepared in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.34-1.52 (m, 2H) 1.78 (m, 2H) 2.14 (m, 1H) 2.43 (m, 4H) 3.32 (d,J=12.30 Hz, 4H) 3.47-3.59 (m, 4H) 5.32 (s, 2H) 6.34 (d, J=8.78 Hz, 1H)7.11 (dd, J=8.93, 2.78 Hz, 1H) 7.47-7.62 (m, 1H). LCMS (ESI) 263 (M+H).

Example 28 Synthesis of 4-[1-(6-nitro-3-pyridyl)-4-piperidyl]thiomorpholine, Compound 28

4-[1-(6-nitro-3-pyridyl)-4-piperidyl] thiomorpholine was synthesized ina manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.40-1.52 (m, 2H) 1.71 (m, 2H) 2.49-2.55 (m, 4H)2.56-2.63 (m, 1H) 2.68-2.75 (m, 4H) 2.88-2.98 (m, 2H) 4.09 (d, J=13.18Hz, 2H) 7.42 (dd, J=9.22, 3.07 Hz, 1H) 8.08 (d, J=9.37 Hz, 1H) 8.20 (d,J=3.22 Hz, 1H).

Example 29 Synthesis of 5-(4-thiomorpholino-1-piperidyl)pyridin-2-amine, Compound 29

5-(4-thiomorpholino-1-piperidyl) pyridin-2-amine was prepared in amanner similar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.47-1.59 (m, 2H) 1.65 (m, 2H) 2.22-2.38 (m, 1H) 2.50-2.59 (m, 6H)2.68-2.82 (m, 4H) 3.33 (d, J=12.00 Hz, 2H) 5.31 (s, 2H) 6.33 (d, J=9.08Hz, 1H) 7.10 (dd, J=8.78, 2.93 Hz, 1H) 7.55 (d, J=2.64 Hz, 1H). LCMS(ESI) 279 (M+H).

Example 30 Synthesis of 2-nitro-5-(1-piperidyl)pyridine, Compound 30

2-nitro-5-(1-piperidyl) pyridine was synthesized in a manner similar tothat used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.56 (m, 6H) 3.49 (d, J=4.39 Hz, 4H) 7.30-7.47 (m, 1H)8.02-8.12 (m, 1H) 8.15-8.26 (m, 1H).

Example 31 Synthesis of 5-(1-piperidyl)pyridin-2-amine, Compound 31

5-(1-piperidyl) pyridin-2-amine was prepared in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.39-1.46 (m, 2H) 1.51-1.62 (m, 4H)2.75-2.92 (m, 4H) 5.30 (s, 2H) 6.34 (d, J=8.78 Hz, 1H) 7.09 (dd, J=8.78,2.93 Hz, 1H) 7.54 (d, J=2.93 Hz, 1H). LCMS (ESI) 178 (M+H).

Example 32 Synthesis of 4-(6-nitro-3-pyridyl) thiomorpholine, Compound32

4-(6-nitro-3-pyridyl) thiomorpholine was synthesized in a manner similarto that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 2.56-2.69 (m, 4H) 3.79-3.92 (m, 4H) 7.43 (dd, J=9.22,3.07 Hz, 1H) 8.10 (d, J=9.37 Hz, 1H) 8.20 (d, J=2.93 Hz, 1H).

Example 33 Synthesis of 5-thiomorpholinopyridin-2-amine, Compound 33

5-thiomorpholinopyridin-2-amine was prepared in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 2.59-2.73 (m, 4H) 3.04-3.20 (m, 4H) 5.41(s, 2H) 6.35 (d, J=8.78 Hz, 1H) 7.10 (dd, J=8.78, 2.93 Hz, 1H) 7.57 (d,J=2.64 Hz, 1H). LCMS (ESI) 196 (M+H).

Example 34 Synthesis of tert-butyl(4R)-5-(6-nitro-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate,Compound 34

tert-butyl(4R)-5-(6-nitro-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylatewas synthesized in a manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.33 (d, J=32.21 Hz, 11H) 1.91 (m, 2H) 3.15 (d, J=10.25Hz, 1H) 3.58 (m, 1H) 4.46 (m, 1H) 4.83 (s, 1H) 7.16 (s, 1H) 7.94 (s, 1H)8.05-8.16 (m, 1H).

Example 35 Synthesis of tert-butyl(4R)-5-(6-amino-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylate,Compound 35

tert-butyl(4R)-5-(6-amino-3-pyridyl)-2,5-diazabicyclo[2.2.1]heptane-2-carboxylatewas prepared in a manner similar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.31 (d, J=31.91 Hz, 11H) 1.83 (m, 2H) 2.71-2.82 (m, 1H) 3.44 (m,1H) 4.30 (d, 2H) 5.08 (s, 2H) 6.35 (d, J=8.78 Hz, 1H) 6.77-6.91 (m, 1H)7.33 (s, 1H). LCMS (ESI) 291 (M+H).

Example 36 Synthesis of N,N-dimethyl-1-(6-nitro-3-pyridyl)piperidin-4-amine, Compound 36

N,N-dimethyl-1-(6-nitro-3-pyridyl)piperidin-4-amine was synthesized in amanner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.30-1.45 (m, 2H) 1.79 (m, 2H) 2.14 (s, 6H) 2.33 (m, 1H)2.92-3.04 (m, 2H) 4.03 (d, J=13.76 Hz, 2H) 7.42 (dd, J=9.22, 3.07 Hz,1H) 8.04-8.11 (m, 1H) 8.21 (d, J=2.93 Hz, 1H).

Example 37 Synthesis of 5-[4-(dimethylamino)-1-piperidyl]pyridin-2-amine, Compound 37

5-[4-(dimethylamino)-1-piperidyl]pyridin-2-amine was prepared in amanner similar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.35-1.50 (m, 2H) 1.69-1.81 (m, 2H) 2.00-2.10 (m, 1H) 2.11-2.22 (s,6H) 3.17-3.36 (m, 4H) 5.19-5.38 (s, 2H) 6.34 (d, J=8.78 Hz, 1H) 7.10(dd, J=8.78, 2.93 Hz, 1H) 7.55 (d, J=2.63 Hz, 1H). LCMS (ESI) 221 (M+H).

Example 38 Synthesis of 4-(6-nitro-3-pyridyl) morpholine, Compound 38

4-(6-nitro-3-pyridyl) morpholine was synthesized in a manner similar tothat used in the synthesis of 2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine.

Example 39 Synthesis of 5-morpholinopyridin-2-amine, Compound 39

5-morpholinopyridin-2-amine was prepared in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl) pyridin-2-amine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 2.91-3.00 (m, 4H) 3.76-3.84 (m, 4H)4.19 (br. s., 2H) 6.45 (d, J=8.78 Hz, 1H) 7.12 (dd, J=8.78, 2.93 Hz, 1H)7.72 (d, J=2.93 Hz, 1H).

Example 40 Synthesis of 5-(4-isobutylpiperazin-1-yl) pyridin-2-amine,Compound 40

1-isobutyl-4-(6-nitro-3-pyridyl)piperazine was synthesized in a mannersimilar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then converted5-(4-isobutylpiperazin-1-yl)pyridin-2-amine in a manner similar to thatused in the synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.88 (d, J=6.73 Hz, 6H) 1.71-1.84(m, 1H) 2.10 (d, J=7.32 Hz, 2H) 2.46-2.58 (m, 4H) 2.97-3.07 (m, 4H) 4.12(s, 2H) 6.45 (d, J=8.78 Hz, 1H) 7.14 (dd, J=8.78, 2.93 Hz, 1H) 7.75 (d,J=2.93 Hz, 1H). LCMS (ESI) 235 (M+H).

Example 41 Synthesis of 5-(4-isopropylpiperazin-1-yl) pyridin-2-amine,Compound 41

1-isopropyl-4-(6-nitro-3-pyridyl)piperazine was synthesized in a mannersimilar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then convertedto 5-(4-isopropylpiperazin-1-yl)pyridin-2-amine in a manner similar tothat used in the synthesis of 5-(4-methylpiperazin-1-yl)pyridin-2-amine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 1.06 (d, J=6.44 Hz, 6H) 2.59-2.75(m, 5H) 2.97-3.10 (m, 4H) 4.13 (s, 2H) 6.45 (d, J=8.78 Hz, 1H) 7.15 (dd,J=9.08, 2.93 Hz, 1H) 7.76 (d, J=2.93 Hz, 1H). LCMS (ESI) 221 (M+H).

Example 42 Synthesis of5-[(2R,6S)-2,6-dimethylmorpholin-4-yl]pyridin-2-amine, Compound 42

(2S,6R)-2,6-dimethyl-4-(6-nitro-3-pyridyl)morpholine was synthesized ina manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then convertedto 5-[(2R,6S)-2,6-dimethylmorpholin-4-yl]pyridin-2-amine in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, CHLOROFORM-d)δ ppm 1.20 (d, J=6.44 Hz, 6H) 2.27-2.39 (m, 2H) 3.11-3.21 (m, 2H)3.70-3.84 (m, 2H) 4.15 (s, 2H) 6.45 (d, J=8.78 Hz, 1H) 7.12 (dd, J=8.78,2.93 Hz, 1H) 7.72 (d, J=2.63 Hz, 1H). LCMS (ESI) 208 (M+H).

Example 43 Synthesis of5-[(3R,5S)-3,5-dimethylpiperazin-1-yl]pyridin-2-amine, Compound 43

(3S,5R)-3,5-dimethyl-1-(6-nitro-3-pyridyl)piperazine was synthesized ina manner similar to that used in the synthesis of2-nitro-5-[4-(1-piperidyl)-1-piperidyl]pyridine which was then convertedto 5-[(3R,5S)-3,5-dimethylpiperazin-1-yl]pyridin-2-amine in a mannersimilar to that used in the synthesis of5-(4-methylpiperazin-1-yl)pyridin-2-amine. ¹HNMR (600 MHz, CHLOROFORM-d)δ ppm 1.09 (d, J=6.44 Hz, 6H) 2.20 (t, J=10.83 Hz, 2H) 2.95-3.08 (m, 2H)3.23 (dd, J=11.71, 2.05 Hz, 2H) 4.13 (s, 2H) 6.45 (d, J=8.78 Hz, 1H)7.14 (dd, J=8.78, 2.93 Hz, 1H) 7.73 (d, J=2.63 Hz, 1H). LCMS (ESI) 207(M+H).

Example 44 Synthesis of Compound 44

tert-butyl N-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate

A solution of intermediate A in ethanol (100 mL) was hydrogenated under30 psi of hydrogen using 10% Pd/C (0.7 g) in a pressure bomb for 7 hrs.After filtration of the reaction mixture through CELITE™, the organiclayer was concentrated under vacuum to afford tert-butylN-(2-amino-3-methyl-butyl) carbamate (3.8 g).

To a solution of 5-bromo-2,4-dichloro-pyrimidine (7.11 g, 0.0312 mole)in ethanol (100 mL) was added diisopropylethyl amine (5.45 mL, 1.0 eq)and tert-butyl N-(2-amino-3-methyl-butyl) carbamate (6.31 g, 0.0312mole). The reaction mixture was stirred at room temperature for 20 hrs.After concentration under vacuum, ethyl acetate and water were added.The organic layer was separated, dried with magnesium sulfate and thenconcentrated under vacuum. The crude product was purified by silica gelcolumn chromatography using hexane/ethyl acetate (0-30%) to affordtert-butyl N-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.77-0.85 (d, J=6.5 Hz, 3H)0.87 (d, J=6.73 Hz, 3H) 1.31-1.39 (m, 9H) 1.82-1.93 (m, 1H) 2.94 (d,J=5.56 Hz, 1H) 3.08-3.22 (m, 2H) 3.98 (d, J=8.20 Hz, 1H) 6.96 (d, J=8.78Hz, 1H) 8.21 (s, 1H). LCMS (ESI) 393 (M+H).

tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-3-methyl-butyl]carbamate

tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-3-methyl-butyl]carbamate was synthesized by hosting tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto Sonogoshira conditions as described for tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamatefollowed by subsequent treatment with TBAF as described in the synthesisof tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.11 (d, J=6.44 Hz, 3H) 1.18 (t, J=7.03Hz, 6H) 1.21-1.26 (m, 12H) 2.88 (br. s., 1H) 3.43-3.78 (m, 6H) 3.97-4.08(m, 1H) 5.61 (s, 1H) 6.65 (s, 1H) 6.71-6.78 (m, 1H) 8.87 (s, 1H). LCMS(ESI) 441 (M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

To a solution tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamatein THF was added TBAF and the contents were heated at reflux for 3 hrs.Ethyl acetate and water were then added and the organic layer separated,dried with magnesium sulfate and then concentrated under vacuum. To thiscrude reaction was added acetic acid/water (9:1) and the contents werestirred for 12 hrs at room temperature. After concentration undervacuum, sat NaHCO₃ and ethyl acetate were added. The organic layer wasseparated, dried and then concentrated under vacuum. The crude reactionproduct thus obtained was dissolved in DMF, oxone was then added and thecontents stirred for 3 hrs. After addition of ethyl acetate, thereaction mixture was filtered through CELITE′ and concentrated undervacuum. Column chromatography of the crude product over silica gel usinghexane/ethyl acetate (0-100%) afforded7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.85 (d, J=7.03 Hz, 3H) 0.97 (d,J=6.73 Hz, 3H) 1.52 (s, 9H) 1.99-2.23 (m, 1H) 3.98 (dd, J=14.05, 3.51Hz, 1H) 4.47-4.71 (m, 2H) 7.47 (s, 1H) 9.17 (s, 1H). LCMS (ESI) 383(M+H).

Compound 44

To7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.050 g, 0.00013 mole) in DCM (1.5 mL) was added DIC (32.7 mg) andDMAP (10 mg). The contents were stirred for 2 hrs. Trifluoroacetic acid(0.4 mL) was then added and stirring continued for an additional 30minutes. After addition of satd NaHCO₃ to neutralize the excess acid,ethyl acetate was added and the organic layer separated, dried usingmagnesium sulfate and then concentrated under vacuum. The crude productwas purified by silica gel column chromatography using hexane/ethylacetate (0-100%) to afford the product. ¹HNMR (600 MHz, DMSO-d₆) δ ppm0.72 (d, J=6.73 Hz, 3H) 0.97 (d, J=6.73 Hz, 3H) 2.09-2.22 (m, 1H) 3.57(dd, J=13.18, 4.98 Hz, 1H) 3.72 (dd, J=13.61, 4.25 Hz, 1H) 4.53 (dd,J=8.05, 3.95 Hz, 1H) 7.20 (s, 1H) 8.34 (d, J=4.98 Hz, 1H) 9.08 (s, 1H).LCMS (ESI) 265 (M+H).

Example 45 Synthesis of Compound 45

Compound 14 was hydrogenated with 10% Pd/C to afford the intermediatetert-butyl N-[(2R)-2-amino-3-methyl-butyl] carbamate, which was thentreated with 5-bromo-2,4-dichloro-pyrimidine using analogous reactionconditions as described for Compound 44 to afford Compound 45 Theanalytical data is consistent with that reported for the racemate(Intermediate 1A).

Example 46 Synthesis of Compound 46

Compound 15 was hydrogenated with 10% Pd/C to afford the intermediatetert-butyl N-[(2S)-2-amino-3-methyl-butyl]carbamate, which was thentreated with 5-bromo-2,4-dichloro-pyrimidine using analogous reactionconditions as described for Compound 44 to afford Compound 46. Theanalytical data (NMR and LCMS) was consistent with that reported for theracemate Compound 44.

Example 47 Synthesis of Compound 47

To a solution of Compound 44 (80 mg, 0.00030 mole) in DMF (3 mL) wasadded a 60% dispersion of sodium hydride in oil (40 mg). After stirringfor 15 minutes, methyl iodide (37 μL, 2 eq) was added. The contents werestirred at room temperature for 30 minutes. Saturated NaHCO₃ was thenadded followed by ethyl acetate. The organic layer was dried withmagnesium sulfate and then concentrated under vacuum to afford theproduct. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.74 (d, J=6.73 Hz, 3H) 0.91 (d,J=6.73 Hz, 3H) 2.04-2.20 (m, 1H) 3.04 (s, 3H) 3.69 (dd, J=13.76, 1.17Hz, 1H) 3.96 (dd, J=13.76, 4.68 Hz, 1H) 4.58 (dd, J=7.32, 3.51 Hz, 1H)7.16 (s, 1H) 9.05 (s, 1H). LCMS (ESI) 279 (M+H).

Example 48 Synthesis of Compound 48

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamate

Compound 18 was hydrogenated with 10% Pd/C in ethanol under a blanket ofhydrogen at 50 psi in a pressure bomb to afford tert-butylN-[(2S)-2-amino-4-methyl-pentyl]carbamate which was then reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.91 (d, J=6.44 Hz, 3H) 0.94 (d,J=6.44 Hz, 3H) 1.32-1.51 (m, 11H) 1.55-1.67 (m, 1H) 3.28 (t, J=5.86 Hz,2H) 4.21-4.42 (m, 1H) 4.84 (s, 1H) 5.84 (d, J=7.32 Hz, 1H) 8.07 (s, 1H).LCMS (ESI) 407 (M+H).

To a solution of tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamate(5.0 g, 12.3 mmole) in tolune (36 mL) and triethylamine (7.2 mL) wasadded under nitrogen, 3,3-diethoxyprop-1-yne (2.8 mL, 19.7 mmole),Pd₂(dba)₃ (1.1 g, 1.23 mmole), and triphenylarsine (3.8 g, 12.3 mmole).The contents were heated to 70 degrees for 24 hrs. After cooling to roomtemperature, the reaction mixture was filtered through CELITE™ and thenconcentrated under vacuum. The crude product was purified by silica gelcolumn chromatography using hexane/ethyl acetate (0-30%) to afford(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.LCMS (ESI) 455 (M+H).

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.88 (d, J=6.44 Hz, 3H) 0.97 (d,J=6.44 Hz, 3H) 1.47 (s, 9H) 1.49-1.54 (m, 1H) 1.56 (t, J=7.17 Hz, 2H)3.98 (dd, J=13.91, 3.07 Hz, 1H) 3.76 (dd, J=13.31, 4.13 Hz, 1H) 4.38 (d,J=14.05 Hz, 1H) 4.90 (t, J=7.17 Hz, 1H) 7.41 (s, 1H) 9.11 (s, 1H). LCMS(M+H) 397.

Compound 48 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.82 (d,J=6.73 Hz, 3H) 0.97 (d, J=6.44 Hz, 3H) 1.34-1.46 (m, 1H) 1.48-1.65 (m,2H) 3.40 (dd, J=13.32, 5.42 Hz, 1H) 3.76 (dd, J=13.47, 4.10 Hz, 1H)4.76-4.92 (m, 1H) 7.17 (s, 1H) 8.34 (d, J=5.27 Hz, 1H) 9.04 (s, 1H).LCMS (ESI) 279 (M+H).

Example 49 Synthesis of Compound 49

Compound 49 was synthesized in a manner similar to that described forCompound 47. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.82 (d, J=6.44 Hz, 3H) 0.97(d, J=6.44 Hz, 3H) 1.37-1.68 (m, 3H) 3.04 (s, 3H) 3.56 (d, J=13.47 Hz,1H) 4.00 (dd, J=13.32, 4.25 Hz, 1H) 4.82-4.94 (m, 1H) 7.16 (s, 1H) 9.03(s, 1H). LCMS (ESI) 293 (M+H).

Example 50 Synthesis of Compound 50

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-pentyl]carbamate

Compound 20 was hydrogenated using 10% Pd/C under hydrogen at 50 psi ina pressure vessel to afford tert-butylN-[(2S)-2-amino-3-methyl-pentyl]carbamate which was reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-pentyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.88-0.95 (m, 6H) 1.11-1.20 (m, 1H)1.34 (s, 9H) 1.44-1.54 (m, 1H) 1.64-1.72 (m, 1H) 3.17-3.27 (m, 1H)3.33-3.43 (m, 1H) 4.11-4.21 (m, 1H) 4.81 (s, 1H) 5.92 (d, J=8.20 Hz, 1H)8.05 (s, 1H). LCMS (ESI) 407.

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3-methyl-pentyl]carbamate

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3-methyl-pentyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.76-0.89 (m, 6H) 1.03 (q, J=7.22 Hz, 3H)1.10-1.17 (m, 3H) 1.25-1.42 (m, 11H) 1.59-1.73 (m, 1H) 3.35-3.47 (m, 4H)3.51-3.73 (m, 2H) 3.99-4.11 (m, 1H) 5.52-5.56 (m, 1H) 6.76-7.03 (m, 2H)8.12-8.23 (m, 1H). LCMS (ESI) 455 (M+H).

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.80 (t, J=7.47 Hz, 3H) 0.86 (d,J=7.03 Hz, 3H) 1.06-1.30 (m, 2H) 1.48 (s, 9H) 1.79-1.96 (m, 1H) 3.95(dd, J=14.05, 3.22 Hz, 1H) 4.52 (d, J=14.35 Hz, 1H) 4.61-4.73 (m, 1H)7.43 (s, 1H) 9.13 (s, 1H). LCMS (ESI) 397 (M+H).

Compound 50 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.74 (t,J=7.32 Hz, 3H) 0.89 (d, J=6.73 Hz, 3H) 1.00-1.12 (m, 2H) 1.82-1.94 (m,1H) 3.55 (dd, J=13.91, 4.83 Hz, 1H) 3.70 (dd, J=13.61, 4.25 Hz, 1H) 4.57(dd, J=7.91, 4.10 Hz, 1H) 7.17 (s, 1H) 8.31 (d, J=5.27 Hz, 1H) 9.05 (s,1H). LCMS (ESI) 279 (M+H).

Example 51 Synthesis of Compound 51

Compound 51 was synthesized in a manner similar to Compound 47. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.77 (t, J=7.47 Hz, 3H) 0.84 (d, J=6.73 Hz, 3H)1.07-1.16 (m, 2H) 1.82-1.95 (m, 1H) 3.03 (s, 3H) 3.68 (d, J=13.76 Hz,1H) 3.96 (dd, J=13.76, 4.39 Hz, 1H) 4.59-4.70 (m, 1H) 7.16 (s, 1H) 9.04(s, 1H). LCMS (ESI) 293 (M+H).

Example 52 Synthesis of Compound 52

tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3,3-dimethyl-butyl]carbamate

Compound 21 was hydrogenated using 10% Pd/C under hydrogen at 50 psi ina pressure vessel to afford tert-butylN-[(2S)-2-amino-3,3-dimethyl-butyl]carbamate which was then reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed using analogous reaction conditions as described fortert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3,3-dimethyl-butyl]carbamate.LCMS (ESI) 407 (M+H).

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3,3-dimethyl-butyl]carbamate

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-3,3-dimethyl-butyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.LCMS (ESI) 455 (M+H).

7-[(1S)-1-[(tert-butoxycarbonylamino)methyl]-2,2-dimethyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1 S)-1-[(tert-butoxycarbonylamino)methyl]-2,2-dimethyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid wassynthesized using the analogous synthetic sequence as that described for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 397 (M+H).

Intermediate 1F was synthesized using an analogous synthetic sequence asthat described for intermediate 1A. LCMS (ESI) 279 (M+H).

Example 53 Synthesis of Compound 53

Compound 53 was synthesized in a manner similar to that described forIntermediate 1CA. LCMS (ESI) 293 (M+H).

Example 54 Synthesis of Compound 54

tert-butylN-[2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-phenyl-ethyl]carbamate

Compound 21 was hydrogenated using 10% Pd/C under hydrogen at 50 psi ina pressure vessel to afford tert-butylN-[(2S)-2-amino-2-phenyl-ethyl]carbamate which was then reacted with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl -butyl]carbamateto afford tert-butylN-[(2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-phenyl-ethyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.32 (s, 9H) 3.29-3.50 (m, 2H) 5.12-5.24(m, 1H) 7.10 (t, J=5.27 Hz, 1H) 7.21 (t, J=6.88 Hz, 1H) 7.26-7.34 (m,4H) 7.89 (d, J=7.32 Hz, 1H) 8.24 (s, 1H). LCMS (ESI) 427 (M+H).

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-phenyl-ethyl]carbamate

tert-butylN-[(2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-phenyl-ethyl]carbamatewas synthesized using similar experimental conditions to those used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.14 (t, J=7.03 Hz, 6H) 1.32 (s, 9H) 3.39(s, 2H) 3.52-3.61 (m, 2H) 3.64-3.73 (m, 2H) 5.17-5.26 (m, 1H) 5.57 (s,1H) 7.07-7.14 (m, 1H) 7.20-7.25 (m, 1H) 7.26-7.33 (m, 4H) 7.90 (d,J=7.61 Hz, 1H) 8.19 (s, 1H). LCMS (ESI) 475 (M+H).

7-[(1S)-2-(tert-butoxycarbonylamino)-1-phenyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1S)-2-(tert-butoxycarbonylamino)-1-phenyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 417 (M+H).

Compound 54

Compound 54 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 3.58-3.69(m, 1H) 4.13 (dd, J=13.47, 4.39 Hz, 1H) 6.07 (d, J=3.81 Hz, 1H) 6.85 (d,J=7.32 Hz, 2H) 7.19-7.31 (m, 3H) 7.34 (s, 1H) 8.27 (d, J=5.27 Hz, 1H)9.13 (s, 1H). LCMS (ESI) 299 (M+H).

Example 55 Synthesis of Compound 55

tert-butylN-[(1S)-1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]-2-methyl-propyl]carbamate

tert-butylN-[(1S)-1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]-2-methyl-propyl]carbamatewas synthesized using 5-bromo-2,4-dichloro-pyrimidine and Intermediate Eusing analogous reaction conditions as described for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.95-1.02 (m, 6H) 1.35-1.45 (m, 9H)1.75-1.90 (m, 1H) 3.35-3.48 (m, 1H) 3.52-3.61 (m, 1H) 3.64-3.76 (m, 1H)4.56 (d, J=8.49 Hz, 1H) 6.47 (s, 1H) 8.07 (s, 1H). LCMS (ESI) 393 (M+H).

tert-butylN-[(1S)-1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]-2-methyl-propyl]carbamate

tert-butylN-[(1S)-1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]-2-methyl-propyl]carbamatewas synthesized using similar experimental conditions to those used inthe synthesis(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.90-1.00 (m, 6H) 1.18-1.25 (m, 6H)1.34-1.36 (m, 9H) 1.69-1.90 (m, 1H) 3.34-3.82 (m, 6H) 4.53-4.77 (m, 1H)5.45-5.55 (m, 1H) 6.37 (dd, J=15.37, 6.59 Hz, 1H) 6.56 (s, 1H) 8.05 (s,1H). LCMS (ESI) 441 (M+H).

7-[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(2S)-2-(tert-butoxycarbonylamino)-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 0.90 (d, J=6.73 Hz, 3H) 0.96(d, J=7.03 Hz, 3H) 1.55-1.66 (m, 10H) 4.14 (dd, J=13.61, 3.95 Hz, 1H)4.52-4.63 (m, 1H) 4.84 (dd, J=13.61, 1.32 Hz, 1H) 7.37 (s, 1H) 8.95 (s,1H). LCMS (ESI) 383 (M+H).

Compound 55

Compound 55 was synthesized using an analogous synthetic sequence asthat described for Compound 44. LCMS (ESI) 265 (M+H).

Example 56 Synthesis of Compound 56

Compound 56 was synthesized using 5-bromo-2,4-dichloro-pyrimidine andCompound 17 as starting materials, and following a similar sequence ofsynthetic steps as for Compound 55. The analytical data was consistentwith that described for its antipode (Compound 55). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.88 (d, J=6.44 Hz, 6H) 1.73-1.86 (m, 1H) 3.67-3.76 (m,2H) 4.11-4.21 (m, 1H) 7.13-7.19 (m, 1H) 8.56 (s, 1H) 9.05 (s, 1H). LCMS(ESI) 265 (M+H).

Example 57 Synthesis of Compound 57

tert-butylN-[2-[5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamate

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewas synthesized using 5-bromo-2,4-dichloro-pyrimidine and tert-butylN-(2-amino-2-methyl-propyl)carbamate using analogous reaction conditionsas described for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.LCMS (ESI) 379 (M+H).

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamate

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewas synthesized using similar experimental conditions to those used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.11-1.22 (m, 6H) 1.31-1.45 (m, 15H)3.10-3.24 (m, 2H) 3.51-3.76 (m, 4H) 5.60 (s, 1H) 6.94 (s, 1H) 7.33 (t,J=6.44 Hz, 1H) 8.18 (s, 1H). LCMS (ESI) 427 (M+H).

7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.43 (s, 9H) 1.73 (s, 6H) 4.06 (s,2H) 7.46 (s, 1H) 9.23 (s, 1H). LCMS (ESI) 369 (M+H).

Compound 57

Compound 57 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.73 (s,6H) 3.50 (d, J=2.93 Hz, 2H) 7.25 (s, 1H) 8.46-8.55 (m, 1H) 9.07 (s, 1H).LCMS (ESI) 251 (M+H).

Example 58 Synthesis of Compound 58

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclohexyl]methyl]carbamate

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclohexyl]methyl]carbamate was synthesized using 5-bromo-2,4-dichloro-pyrimidine andIntermediate K using the analogous reaction conditions as described fortert-butyl N-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.18-1.54 (m, 17H) 2.23 (d,J=14.35 Hz, 2H) 3.36 (d, J=6.44 Hz, 2H) 5.82 (s, 1H) 6.93 (s, 1H) 8.22(s, 1H). LCMS (ESI) 419 (M+H).

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclohexyl]methyl] carbamate

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclohexyl]methyl]carbamate was synthesized using similar experimental conditions to thoseused in the synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.08-1.16 (m, 6H) 1.17-1.54 (m, 17H) 2.13(br. s., 2H) 3.36 (d, J=6.73 Hz, 2H) 3.50-3.69 (m, 4H) 5.72 (s, 1H) 6.94(s, 1H) 5.72 (br. s., 1H) 8.17 (s, 1H). LCMS (ESI) 467 (M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]cyclohexyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[1-[(tert-butoxycarbonylamino)methyl]cyclohexyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid wassynthesized using analogous synthetic sequence as that described for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylic acid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.37-1.54(m, 13H) 1.75 (br. s., 4H) 2.74 (br. s., 2H) 3.78-3.84 (m, 2H) 7.44-7.51(m, 1H) 8.23 (s, 1H) 9.11 (s, 1H). LCMS (ESI) 409 (M+H).

Compound 58

Compound 58 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.28 (br.s., 2H) 1.42 (br. s., 2H) 1.70 (br. s., 4H) 1.85-1.95 (m, 2H) 2.69 (m,2H) 7.16-7.25 (m, 1H) 8.41 (br. s., 1H) 9.04 (s, 1H). LCMS 291 (M+H).

Example 59 Synthesis of Compound 59

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamate

tert-butyl N-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl] carbamate was synthesized using 5-bromo-2,4-dichloro-pyrimidineand Intermediate L using analogous reaction conditions as described fortert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.34 (s, 9H) 1.50-1.58 (m, 2H) 1.63-1.78(m, 4H) 1.96-2.06 (m, 2H) 3.25 (d, J=6.15 Hz, 2H) 6.71 (s, 1H) 7.18 (t,J=6.29 Hz, 1H) 8.20 (s, 1H). LCMS (ESI) 405 (M+H).

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]methyl] carbamate

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]methyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.LCMS (ESI) 453 (M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[1-[(tert-butoxycarbonylamino)methyl]cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.47 (s, 9H) 1.74 (br. s., 2H) 1.88(br. s., 2H) 2.04 (br. s., 2H) 2.41-2.45 (m, 2H) 4.06 (s, 2H) 7.45 (s,1H) 9.11 (s, 1H). LCMS (ESI) 395 (M+H).

Compound 59

Compound 59 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.72 (br.s., 2H) 1.86-1.93 (m, 2H) 1.99 (d, J=3.81 Hz, 2H) 2.40 (br. s., 2H) 3.48(d, J=2.34 Hz, 2H) 7.22 (s, 1H) 8.53 (br. s., 1H) 9.05 (s, 1H). LCMS(ESI) 277 (M+H).

Example 60 Synthesis of Compound 60

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl] carbamate

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-4-methyl-pentyl]carbamatewas synthesized using 5-bromo-2,4-dichloro-pyrimidine and Intermediate Busing analogous reaction conditions as described for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.The analytical data is consistent with that described for theL-enantiomer.

tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-4-methyl-pentyl]carbamate

tert-butyl N-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-4-methyl-pentyl]carbamate was synthesized using similarexperimental conditions to that used in the synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate.¹HNMR (600 MHz, CHLOROFORM-d) δ ppm 1.21-1.31 (m, 12H) 1.38-1.46 (m,11H) 1.70 (m, 1H) 3.24 (m, 2H) 3.65-3.82 (m, 4H) 4.86 (br s., 1H), 5.65(s, 1H) 5.85 (br s., 1H) 6.94 (s, 1H) 8.21 (s, 1H). LCMS (ESI) 455(M+H).

7-[1-[(tert-butoxycarbonylamino)methyl]-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[1-[(tert-butoxycarbonylamino)methyl]-3-methyl-butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. The analytical data was consistent with that described for theL-isomer.

Compound 60

Compound 60 was synthesized using an analogous synthetic sequence asthat described for Compound 44. The analytical data was consistent withthat described for the L-isomer.

Example 61 Synthesis of Compound 61

To a solution of Compound 60 (100 mg, 0.00024 mole) in DMF (3.0 mL) wasadded sodium hydride (60% dispersion in oil), (27.6 mg, 3 eq). Afterstirring for 15 mins, methyl iodide (30, 2 eq) was added. The contentswere stirred at room temperature for 30 mins. After the addition of satNaHCO₃, ethyl acetate was added. Separation of the organic layerfollowed by drying with magnesium sulfate and concentration under vacuumafforded the product. Analytical data was similar to the Compound 49.

Example 62 Synthesis of Compound 62

tert-butylN-[(1S,2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]carbamate

tert-butylN-[(1S,2S)-2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]carbamatewas synthesized by treating tert-butylN-[(1S,2S)-2-aminocyclopentyl]carbamate with5-bromo-2,4-dichloro-pyrimidine using analogous reaction conditions asdescribed for tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-3-methyl-butyl]carbamate.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.27 (s, 9H) 1.42-1.54 (m, 2H) 1.56-1.65(m, 2H) 1.80-1.88 (m, 1H) 1.96-2.01 (m, 1H) 3.88-3.96 (m, 1H) 4.03-4.09(m, 1H) 6.91 (d, J=8.20 Hz, 1H) 7.41 (d, J=7.32 Hz, 1H) 8.18 (s, 1H).LCMS (ESI) 391 (M+H).

tert-butylN-[(1S,2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]carbamate

tert-butylN-[(1S,2S)-2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]carbamatewas synthesized using similar experimental conditions to that used inthe synthesis of(2S)—N2-[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]-4-methyl-pentane-1,2-diamine.¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.13 (t, 6H) 1.28 (s, 9H) 1.42-1.52 (m,2H) 1.58-1.65 (m, 2H) 1.81-1.90 (m, 1H) 1.99-2.08 (m, 1H) 3.49-3.60 (m,2H) 3.63-3.71 (m, 2H) 3.84-3.93 (m, 1H) 3.96-4.04 (m, 1H) 5.53 (s, 1H)6.96 (d, J=7.90 Hz, 1H) 7.34 (d, J=7.03 Hz, 1H) 8.14 (s, 1H). LCMS (ESI)439 (M+H).

7-[(1S,2S)-2-(tert-butoxycarbonylamino)cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid

7-[(1 S,2S)-2-(tert-butoxycarbonylamino)cyclopentyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using the analogous synthetic sequence as thatdescribed for7-[1-[(tert-butoxycarbonylamino)methyl]-2-methyl-propyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.41-1.52 (m, 9H) 1.55-1.68 (m, 1H)1.88-2.00 (m, 2H) 2.05-2.15 (m, 1H) 2.26-2.35 (m, 1H) 2.71-2.89 (m, 1H)4.01-4.16 (m, 1H) 4.28-4.45 (m, 1H) 7.41 (s, 1H) 9.11 (s, 1H). LCMS(ESI) 381 (M+H).

Compound 62

Compound 62 was synthesized using an analogous synthetic sequence asthat described for Compound 44. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.48-1.60(m, 1H) 1.88-1.98 (m, 3H) 1.99-2.08 (m, 1H) 2.66-2.75 (m, 1H) 3.63-3.74(m, 1H) 3.99-4.12 (m, 1H) 7.21 (s, 1H) 8.89 (s, 1H) 9.04 (s, 1H). LCMS(ESI) 263 (M+H).

Example 63 Synthesis of Compound 63

To chloro tricycliclactam (0.050 g, 0.225 mmole) in dioxane (2.0 mL)under nitrogen was added 5-(4-methylpiperazin-1-yl)pyridin-2-amine(0.052 g, 1.2 eq, 0.270 mmole) followed by the addition of Pd₂(dba)3(18.5 mg), BINAP (25 mg) and sodium-tert-butoxide (31 mg, 0.324 mmole).The contents of the flask are degassed for 10 minutes and then heated to100 degrees for 12 hours. The crude reaction was loaded on a silica gelcolumn and eluted with DCM/MeOH (0-15%) to afford the desired product(26 mg). To this compound dissolved in DCM/MeOH (10%) was added 3N HClin iso-propanol (2 eq) and the reaction was stirred overnight.Concentration under vacuum afforded the hydrochloride salt. ¹HNMR(d₆-DMSO) δ ppm 11.13 (brs, 1H), 9.07 (s, 1H), 8.42 (s, 1H), 8.03 (br m1H), 7.99 (s, 1H), 7.67 (brm, 1H), 7.18 (s, 1H), 4.33 (m, 2H), 3.79 (m,2H), 3.64 (m, 2H), 3.50 (m, 2H), 3.16 (m, 4H), 2.79 (s, 3H). LCMS (ESI)379 (M+H).

Example 64 Synthesis of Compound 64

To chloro tricycliclactam (0.075 g, 0.338 mmole) in dioxane (3.5 mL)under nitrogen was added tert-butyl4-(6-amino-3-pyridyl)piperazine-1-carboxylate (0.098 g, 1.05 eq)followed by the addition of Pd₂(dba)3 (27 mg), BINAP (36 mg) andsodium-tert-butoxide (45 mg). The contents were heated at reflux for 11hrs. The crude reaction was loaded onto a silica gel column and elutedwith DCM/MeOH (0-10%) to afford the desired product (32 mg). ¹HNMR(d₆-DMSO) δ ppm 9.48 (s, 1H), 8.84 (s, 1H), 8.29 (s, 1H), 8.18 (s, 1H),7.99 (s, 1H), 7.42 (m, 1H), 6.98 (s, 1H), 4.23 (m, 2H), 3.59 (m, 2H),3.45 (m, 4H), 3.50 (m, 2H), 3.05 (m, 4H). LCMS (ESI) 465 (M+H).

Example 65 Synthesis of Compound 65

To a solution of Compound 64 (23 mg) in 10% DCM/MeOH was added 10 mL ofa 3M solution of HCl in iso-propanol. The contents were stirred for 16hrs. Concentration of the reaction mixture afforded the hydrochloridesalt. ¹HNMR (d₆-DMSO) δ ppm 9.01 (s, 1H), 7.94 (m, 1H), 7.86 (m, 1H),7.23 (s, 1H), 4.30 (m, 2H), 3.64 (m, 2H), 3.36 (m, 4H), 3.25 (m, 4H).LCMS (ESI) 465 (M+H).

Example 66 Synthesis of Compound 66

To chloro-N-methyltricyclic amide (0.080 g, 0.338 mmole) in dioxane (3.5mL) under nitrogen was added tert-butyl4-(6-amino-3-pyridyl)piperazine-1-carboxylate 0.102 g (1.1 eq) followedby the addition of Pd₂(dba)3 (27 mg), BINAP (36 mg) andsodium-tert-butoxide (45 mg). The contents were heated at reflux for 11hrs. The crude product was purified using silica gel columnchromatography with an eluent of dichloromethane/methanol (0-5%) toafford the desired product (44 mg). ¹HNMR (d₆-DMSO) δ ppm 9.49 (s, 1H),8.85 (s, 1H), 8.32 (m, 1H), 8.02 (s, 1H), 7.44 (m, 1H), 7.00 (s, 1H),4.33 (m, 2H), 3.80 (m, 2H), 3.48 (m, 4H), 3.07 (m, 4H), 3.05 (s, 3H),1.42 (s, 9H). LCMS (ESI) 479 (M+H).

Example 67 Synthesis of Compound 67

To Compound 66 (32 mg) was added 3N HCL (10 mL) in isopropanol and thecontents were stirred at room temperature overnight for 16 hrs.Concentration afforded the hydrochloride salt. ¹HNMR (d₆-DMSO) δ ppm9.13 (m, 2H), 8.11 (m, 1H), 8.10 (s, 1H), 7.62 (m, 1H), 7.21 (s, 1H),4.43 (m, 2H), 3.85 (m, 2H), 3.41 (m, 4H), 3.28 (m, 4H), 3.08 (s, 3H).LCMS (ESI) 379 (M+H).

Example 68 Synthesis of Compound 68

Compound 68 was synthesized using similar experimental conditions tothat described for compound 64. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.79 (d,J=7.03 Hz, 3H) 1.01 (d, J=6.73 Hz, 3H) 1.35-1.48 (m, 9H) 2.16 (dd,J=14.64, 6.73 Hz, 1H) 3.00-3.14 (m, 4H) 3.40-3.51 (m, 4H) 3.51-3.60 (m,1H) 3.63-3.74 (m, 1H) 4.44 (dd, J=7.90, 3.81 Hz, 1H) 6.99 (s, 1H) 7.46(dd, J=8.93, 2.78 Hz, 1H) 7.94-8.09 (m, 2H) 8.31 (dd, J=9.08, 1.46 Hz,1H) 8.85 (s, 1H) 9.46 (s, 1H). LCMS (ESI) 507 (M+H).

Example 69 Synthesis of Compound 69

Compound 69 was synthesized using similar experimental conditions tothose described for compound 63 and was recovered as an HCl salt. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.77-0.86 (m, 3H) 0.96 (d, J=7.03 Hz, 3H)2.10-2.24 (m, 1H) 3.07 (s, 3H) 3.37-3.79 (m, 8H) 4.00 (dd, J=13.61, 4.54Hz, 2H) 4.63-4.73 (m, 1H) 7.20 (s, 1H) 7.58-7.71 (m, 1H) 7.99 (d, J=2.34Hz, 1H) 8.12 (d, J=9.37 Hz, 1H) 9.11 (s, 1H) 9.41 (br. s., 2H) 11.76(br. s., 1H). LCMS (ESI) 421 (M+H).

Example 70 Synthesis of Compound 70

Compound 70 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. The characterization data (NMR and LCMS) was consistent with thatreported for compound 71.

Example 71 Synthesis of Compound 71

Compound 71 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.79 (d, J=6.73 Hz, 3H) 1.01 (d,J=6.73 Hz, 3H) 2.18 (dd, J=14.49, 7.17 Hz, 1H) 3.18-3.84 (m, 10H)4.53-4.71 (m, 1H) 7.24 (s, 1H) 7.65 (d, J=9.37 Hz, 1H) 8.01 (d, J=2.64Hz, 1H) 8.14 (d, J=1.46 Hz, 1H) 8.35 (d, J=5.27 Hz, 1H) 9.14 (s, 1H)9.46 (s, 2H) 11.80 (s, 1H) LCMS (ESI) 407 (M+H).

Example 72 Synthesis of Compound 72 (Compound UUU)

Compound 72 was synthesized using similar experimental conditions tothat described for compounds 64 and 65 and was recovered as an HCl salt.¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.77 (d, J=7.03 Hz, 3H) 0.99 (d, J=6.73Hz, 3H) 2.10-2.24 (m, 1H) 3.18-3.81 (m, 10H) 4.54-4.69 (m, 1H) 7.22 (s,1H) 7.63 (d, J=9.08 Hz, 1H) 7.99 (d, J=2.63 Hz, 1H) 8.11 (s, 1H) 8.33(d, J=5.27 Hz, 1H) 9.12 (s, 1H) 9.43 (s, 2H) 11.77 (s, 1H). LCMS (ESI)407 (M+H).

Example 73 Synthesis of Compound 73

Compound 73 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹H NMR (600 MHz, DMSO-d₆) δ ppm 0.84 (d, J=6.73 Hz, 3H) 0.98 (d,J=6.73 Hz, 3H) 2.12-2.26 (m, 1H) 3.09 (s, 3H) 3.22-3.81 (m, 8H) 4.01(dd, J=13.61, 4.25 Hz, 2H) 4.59-4.72 (m, 1H) 7.19 (s, 1H) 7.74 (s, 1H)7.96-8.10 (m, 2H) 9.08 (s, 1H) 9.22 (s, 2H). LCMS (ESI) 421 (M+H).

Example 74 Synthesis of Compound 74

Compound 74 was synthesized using similar experimental conditions tothose described for compound 63 and was recovered as an HCl salt. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.85 (d, J=4.98 Hz, 3H) 0.95 (d, J=4.98 Hz, 3H)1.42-1.70 (m, 3H) 2.77 (d, J=2.93 Hz, 3H) 3.07-4.14 (m, 10H) 4.95 (s,1H) 7.20 (s, 1H) 7.66 (d, J=9.66 Hz, 1H) 7.94 (s, 1H) 8.08-8.16 (m, 1H)8.33 (d, J=4.68 Hz, 1H) 9.09 (s, 1H) 11.38 (s, 1H) 11.71 (s, 1H). LCMS(ESI) 435 (M+H).

Example 75 Synthesis of Compound 75

Compound 75 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹H NMR (600 MHz, DMSO-d₆) δ ppm 0.87 (d, J=6.15 Hz, 3H) 0.94 (d,J=6.15 Hz, 3H) 1.57 (d, J=84.61 Hz, 3H) 3.05 (s, 3H) 3.13-3.55 (m, 8H)3.69 (d, J=78.17 Hz, 2H) 4.90 (s, 1H) 7.15 (s, 1H) 7.63-7.85 (m, 1H)7.93 (s, 1H) 8.26 (s, 1H) 9.03 (s, 1H) 9.20 (s, 2H). LCMS (ESI) 421(M+H).

Example 76 Synthesis of Compound 76

Compound 76 was synthesized using similar experimental conditions tothose described for compound 63 and was recovered as an HCl salt. ¹HNMR(600 MHz, DMSO-d₆) δ ppm 0.85 (d, J=6.44 Hz, 3H) 0.95 (d, J=6.44 Hz, 3H)1.43-1.70 (m, 3H) 2.78 (d, J=2.93 Hz, 3H) 3.05 (s, 3H) 3.24-3.84 (m, 8H)4.01 (d, J=9.66 Hz, 2H) 4.89-5.01 (m, 1H) 7.15 (s, 1H) 7.77 (s, 1H)7.91-8.05 (m, 2H) 9.03 (s, 1H) 10.96-11.55 (m, 2H). LCMS (ESI) 449(M+H).

Example 77 Synthesis of Compound 77

Compound 77 was synthesized using similar experimental conditions tothose described for compounds 64 and 65 and was recovered as an HClsalt. ¹H NMR (600 MHz, DMSO-d₆) δ ppm 0.83-0.88 (d, J=6.15 Hz, 3H) 0.95(d, J=6.15 Hz, 3H) 1.40-1.71 (m, 3H) 3.28-3.83 (m, 8H) 4.00 (d, J=3.22Hz, 2H) 4.91-5.08 (m, 1H) 7.17 (s, 1H) 7.68 (d, J=9.66 Hz, 1H) 7.93 (s,1H) 8.07 (s, 1H) 9.06 (s, 1H) 9.40 (s, 2H) 11.59 (s, 1H). LCMS (ESI) 435(M+H).

Example 78 Synthesis of Compound 78

To Compound 50 0.060 g (0.205 mmole) was added5-(4-methylpiperazin-1-yl)pyridin-2-amine (35.42 mg, 0.9 eq) followed bythe addition of 1,4-dioxane (3 mL). After degassing with nitrogen,Pd₂dba₃ (12 mg), BINAP (16 mg) and sodium tert-butoxide (24 mg) wereadded. The contents were then heated at 90 degrees in a CEM Discoverymicrowave for 3 hrs. The reaction was then loaded onto a silica gelcolumn and purified by eluting with DCM/MeOH (0-15%). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.75 (t, J=7.47 Hz, 3H) 0.91 (d, J=6.73 Hz, 3H) 1.04-1.20(m, 2H) 1.80-1.98 (m, 1H) 2.77 (d, J=3.81 Hz, 3H) 2.94-3.90 (m, 10H)4.54-4.68 (m, 1H) 7.06-7.23 (m, 2H) 7.56-7.75 (m, 1H) 7.90-8.12 (m, 2H)8.29 (s, 1H) 9.07 (s, 1H) 10.98-11.74 (m, 2H). LCMS (ESI) 435 (M+H).

Example 79 Synthesis of Compound 79

Compound 79 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.75(t, J=7.32 Hz, 3H) 0.90 (d, J=6.73 Hz, 3H) 1.07-1.15 (m, 2H) 1.85-1.94(m, 1H) 3.17-3.75 (m, 10H) 4.58-4.67 (m, 1H) 7.17 (s, 1H) 7.71 (s, 1H)7.96 (s, 1H) 7.98-8.05 (m, 1H) 8.28 (d, J=4.10 Hz, 1H) 9.06 (s, 1H) 9.39(s, 2H). LCMS (ESI) 421 (M+H).

Example 80 Synthesis of Compound 80

Compound 80 was synthesized in a similar manner to that described forcompound 78. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.78 (t, J=7.32 Hz, 3H) 0.86(d, J=6.73 Hz, 3H) 1.13-1.21 (m, 2H) 1.84-1.96 (m, 1H) 2.77 (d, J=4.39Hz, 3H) 3.04 (s, 3H) 3.11-3.84 (m, 8H) 3.98 (dd, J=13.61, 4.25 Hz, 2H)4.66-4.74 (m, 1H) 7.17 (s, 1H) 7.64 (s, 1H) 7.96 (d, J=2.34 Hz, 1H)8.03-8.13 (m, 1H) 9.08 (s, 1H) 11.26 (s, 1H) 11.66 (s, 1H). LCMS (ESI)449 (M+H).

Example 81 Synthesis of Compound 81

The compound was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.78(t, J=7.32 Hz, 3H) 0.85 (d, J=6.73 Hz, 3H) 1.10-1.27 (m, 2H) 1.82-1.99(m, 1H) 3.04 (s, 3H) 3.28-3.77 (m, 8H) 3.97 (dd, J=13.91, 4.54 Hz, 2H)4.62-4.75 (m, 1H) 7.07-7.24 (m, 1H) 7.62-7.75 (m, 1H) 7.94 (d, J=2.34Hz, 1H) 7.97-8.08 (m, 1H) 9.05 (s, 1H) 9.29 (s, 2H). LCMS (ESI) 435(M+H).

Example 82 Synthesis of Compound 82

The compound was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.96(s, 9H) 3.15-3.87 (m, 10H) 4.42-4.53 (m, 1H) 6.99 (s, 1H) 7.24 (s, 1H)8.06 (s, 1H) 8.11-8.21 (m, 1H) 8.79-8.98 (m, 2H) 9.25 (s, 2H) 9.88 (s,1H). LCMS (ESI) 421 (M+H).

Example 83 Synthesis of Compound 83

Compound 83 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.95(s, 9H) 2.79 (d, J=4.10 Hz, 3H) 3.06-3.86 (m, 10H) 4.56-4.67 (m, 1H)7.17 (s, 1H) 7.70 (s, 1H) 7.96 (d, J=2.63 Hz, 1H) 7.99-8.08 (m, 1H) 8.26(s, 1H) 9.06 (s, 1H) 10.80 (s, 1H). LCMS (ESI) 435 (M+H).

Example 84 Synthesis of Compound 84

Compound 84 was synthesized in a similar manner to that described forcompound 78 and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δppm 2.75-2.81 (m, 3H) 3.12-3.16 (m, 2H) 3.46-3.54 (m, 4H) 3.60-3.69 (m,2H) 3.72-3.79 (m, 1H) 4.07-4.18 (m, 2H) 6.06-6.09 (m, 1H) 6.90 (d,J=7.61 Hz, 2H) 7.20-7.31 (m, 3H) 7.33 (s, 1H) 7.49-7.55 (m, 1H)7.62-7.70 (m, 1H) 7.92 (d, J=2.93 Hz, 1H) 8.22 (s, 1H) 9.14 (s, 1H).LCMS (ESI) 455 (M+H).

Example 85 Synthesis of Compound 85

Compound 85 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 3.21(s, 4H) 3.35-3.67 (m, 5H) 4.07-4.20 (m, 2H) 6.13 (s, 1H) 6.90 (d, J=7.32Hz, 2H) 7.22-7.31 (m, 3H) 7.36 (s, 1H) 7.48 (d, J=9.37 Hz, 1H) 7.93 (d,J=2.34 Hz, 1H) 8.04-8.11 (m, 1H) 8.25 (d, J=4.98 Hz, 1H) 9.17 (s, 1H)11.77 (br, s., 1H). LCMS (ESI) 441 (M+H).

Example 86 Synthesis of Compound 86

Compound 86 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 0.90(d, J=6.15 Hz, 6H) 1.72-1.89 (m, 1H) 3.15-3.92 (m, 9H) 4.10-4.46 (m, 2H)7.18 (s, 1H) 7.59 (d, J=8.78 Hz, 1H) 8.00 (s, 1H) 8.13 (d, J=9.37 Hz,1H) 8.55 (s, 1H) 9.09 (s, 1H) 9.67 (s, 2H) 11.91 (s, 1H). LCMS (ESI) 407(ESI).

Example 87 Synthesis of Compound 87

Compound 87 was synthesized in a manner similar to compound 86 and wasconverted to an HCl salt. The characterization data (NMR and LCMS) wassimilar to that obtained for the antipode compound 86.

Example 88 Synthesis of Compound 88

Compound 88 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.78(s, 6H) 3.40-3.53 (m, 6H) 3.64-3.73 (m, 4H) 7.27 (s, 1H) 7.66 (d, J=9.37Hz, 1H) 7.98 (d, J=2.34 Hz, 1H) 8.12 (br. s., 1H) 8.47 (br. s., 1H) 9.11(s, 1H) 9.45 (br. s., 2H) 11.62 (br. s., 1H). LCMS (ESI) 393 (M+H).

Example 89 Synthesis of Compound 89 (Also Referred to as Compound T)

Compound 89 was synthesized in a similar manner to that described forcompound 78 and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δppm 1.47 (br. s., 6H) 1.72 (br. s., 2H) 1.92 (br. s., 2H) 2.77 (br. s.,3H) 3.18 (br. s., 2H) 3.46 (br. s., 2H) 3.63 (br. s., 2H) 3.66 (d,J=6.15 Hz, 2H) 3.80 (br. s., 2H) 7.25 (s, 1H) 7.63 (br. s., 2H) 7.94(br. s., 1H) 8.10 (br. s., 1H) 8.39 (br. s., 1H) 9.08 (br. s., 1H) 11.59(br. s., 1H). LCMS (ESI) 447 (M+H).

Example 90 Synthesis of Compound 90 (Also Referred to as Compound Q)

Compound 90 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.27-1.64 (m, 6H) 1.71 (br. s., 2H) 1.91 (br. s., 2H) 2.80 (br. s., 1H)3.17-3.24 (m, 2H) 3.41 (br. s., 4H) 3.65 (br. s., 4H) 7.26 (br. s., 1H)7.63 (br. s., 1 H) 7.94 (br. s., 1H) 8.13 (br. s., 1H) 8.40 (br. s., 1H)9.09 (br. s., 1H) 9.62 (br. s., 1H) 11.71 (br. s., 1H). LCMS (ESI) 433(M+H).

Example 91 Synthesis of Compound 91 (Also Referred to as Compound ZZ)

Compound 91 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.64-1.75 (m, 2H) 1.83-1.92 (m, 2H) 1.96-2.06 (m, 2H)2.49-2.58 (m, 2H) 2.79 (d, J=3.81 Hz, 3H) 3.06-3.18 (m, 4H) 3.59-3.69(m, 2H) 3.73-3.83 (m, 2H) 4.04-4.12 (m, 2H) 7.17 (br. s., 1H) 7.60-7.70(m, 2H) 7.70-7.92 (m, 2H) 7.96 (br. s., 1H) 8.41 (br. s., 1H) 8.98 (br.s., 1H) 10.77 (br. s., 1H). LCMS (ESI) 433 (M+H).

Example 92 Synthesis of Compound 92

Compound 92 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.64-1.75 (m, 2H) 1.84-1.92 (m, 2H) 1.96-2.05 (m, 2H) 2.48-2.56 (m, 2H)3.22 (br. s., 4H) 3.42-3.48 (m, 4H) 3.60-3.69 (m, 2H) 4.05-4.13 (m, 1H)7.18 (s, 1H) 7.65 (d, J=13.47 Hz, 1H) 7.70-7.77 (m, 1H) 7.94 (d, J=1.76Hz, 1H) 8.42 (br. s., 1H) 9.00 (s, 1H) 9.15 (br. s., 2H). LCMS (ESI) 419(M+H).

Example 93 Synthesis of Compound 93

Compound 93 was synthesized in a similar manner to that described forcompound 78 followed by the deblocking step described for compound 65and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm 1.76(br. s., 2H) 1.89 (br. s., 2H) 2.03 (br. s., 2H) 2.47-2.58 (m, 2H) 3.04(s, 3H) 3.22 (br. s., 4H) 3.39 (br. s., 4H) 3.66 (s, 2H) 7.21 (s, 1H)7.67 (d, J=9.37 Hz, 1H) 7.93 (br. s., 1H) 7.98-8.09 (m, 1H) 9.04 (s, 1H)9.34 (br. s., 2H) 11.31 (br. s., 1H). LCMS (ESI) 433 (M+H).

Example 94 Synthesis of Compound 94

Compound 94 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.66-1.77 (m, 2H) 1.84-1.94 (m, 2H) 1.96-2.08 (m, 2H)2.48-2.57 (m, 2H) 3.36-3.52 (m, 4H) 3.60-3.80 (m, 6H) 7.21 (s, 1H)7.53-7.74 (m, 2H) 7.86 (s, 1H) 8.02 (s, 1H) 8.45 (s, 1H) 9.03 (s, 1H)11.19 (br. s., 1H). LCMS (ESI) 420 (M+H).

Example 95 Synthesis of Compound 95

Compound 95 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.65-1.79 (m, 2H) 1.85-1.95 (m, 2H) 1.97-2.08 (m, 2H)2.47-2.54 (m, 2H) 3.40-3.58 (m, 5H) 3.65 (dd, J=21.67, 5.56 Hz, 1H)3.69-3.78 (m, 4H) 7.24 (s, 1H) 7.97-8.17 (m, 2H) 8.48 (s, 1H) 9.08 (s,1H) 11.81 (s, 1H). LCMS (ESI) 421 (M+H).

Example 96 Synthesis of Compound 96

Compound 96 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.55-1.74 (m, 2H) 1.80-1.98 (m, 4H) 2.48-2.60 (m, 2H)3.40-3.50 (m, 4H) 3.57-3.72 (m, 2H) 3.90-4.20 (m, 4H) 7.08 (s, 1H)7.37-7.57 (m, 2H) 7.70 (m, 2H) 8.32 (s, 1H) 8.88 (s, 1H) 9.98 (s, 1H).LCMS (ESI) 419 (M+H).

Example 97 Synthesis of Compound 97 (Also Referred to as Compound III)

Compound 97 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.30 (d, J=5.27 Hz, 6H) 1.65-1.78 (m, 2H) 1.83-1.95 (m,2H) 1.97-2.10 (m, 2H) 2.45-2.55 (m, 2H) 3.25-3.36 (m, 1H) 3.39-3.48 (m,4H) 3.60-3.70 (m, 4H) 3.75-4.15 (m, 2H) 7.24 (s, 1H) 7.54-7.75 (m, 2H)7.95 (s, 1H) 8.10 (s, 1H) 8.49 (s, 1H) 9.07 (s, 1H) 11.25 (s, 1H) 11.48(s, 1H). LCMS (ESI) 461 (M+H).

Example 98 Synthesis of Compound 98

Compound 98 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹H NMR (600 MHz,DMSO-d₆) δ ppm 0.99 (d, J=6.15 Hz, 6H) 1.65-1.78 (m, 2H) 1.90 (m, 2H)1.97-2.08 (m, 2H) 2.08-2.17 (m, 1H) 2.45-2.55 (m, 2H) 2.88-3.02 (m, 2H)3.33-3.48 (m, 4H) 3.50-3.90 (m, 6H) 7.24 (s, 1H) 7.67 (s, 2H) 7.94 (s,1H) 8.12 (s, 1H) 8.49 (s, 1H) 9.07 (s, 1H) 10.77 (s, 1H) 11.51 (s, 1H).LCMS (ESI) 475 (M+H).

Example 99 Synthesis of Compound 99

Compound 99 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.13 (d, J=5.86 Hz, 6H) 1.66-1.77 (m, 2H) 1.84-1.94 (m,2H) 1.97-2.09 (m, 2H) 2.40-2.53 (m, 2H) 3.37-3.49 (m, 2H) 3.50-3.59 (m,2H) 3.59-3.73 (m, 4H) 7.23 (s, 1H) 7.64 (m, 3H) 7.85 (s, 1H) 8.11 (s,1H) 8.47 (s, 1H) 9.05 (s, 1H). 11.35 (br s., 1H). LCMS (ESI) 448 (M+H).

Example 100 Synthesis of Compound 100

Compound 100 was synthesized using similar conditions to that describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.50-1.57 (m, 2H) 1.62-1.68 (m, 3H) 1.68-1.75 (m, 2H)1.84-1.92 (m, 2H) 1.97-2.08 (m, 2H) 2.48-2.53 (m, 2H) 3.14-3.23 (m, 4H)3.43-3.47 (m, 2H) 3.58-3.70 (m, 2H) 7.22 (s, 1H) 7.58-7.70 (m, 2H)7.85-8.00 (m, 1H) 8.16 (d, 1H) 8.46 (s, 1H) 9.04 (s, 1H) 11.37 (br s.,1H). LCMS (ESI) 418 (M+H).

Example 101 Synthesis of Compound 101 (Also Referred to as Compound WW)

Compound 101 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.72 (s, 2H) 1.90 (s, 4H) 2.03 (s, 2H) 2.21 (s, 2H)2.48-2.54 (m, 2H) 2.73 (s, 2H) 3.03 (s, 2H) 3.25-3.35 (m, 1H) 3.38-3.48(m, 4H) 3.65-3.99 (m, 5H) 7.23 (s, 1H) 7.63 (d, J=9.66 Hz, 1H) 7.90 (s,1H) 8.13 (s, 1H) 8.47 (s, 1H) 9.06 (s, 1H) 10.50 (br s., 1H). LCMS (ESI)503 (M+H).

Example 102 Synthesis of Compound 102 (Also Referred to as Compound HHH)

Compound 102 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.63-1.85 (m, 6H) 1.87-1.92 (m, 2H) 1.99-2.06 (m, 2H)2.15-2.23 (m, 2H) 2.47-2.53 (m, 1H) 2.69-2.79 (m, 2H) 2.81-2.91 (m, 2H)2.98-3.08 (m, 2H) 3.32-3.48 (m, 4H) 3.57-3.72 (m, 4H) 3.77-3.85 (m, 2H)7.22 (s, 1H) 7.60-7.68 (m, 2H) 7.90 (s, 1H) 8.07 (s, 1H) 8.46 (s, 1H)9.04 (s, 1H). 11.41 (br s., 1H). LCMS (ESI) 501 (M+H).

Example 103 Synthesis of Compound 103

Compound 103 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.64-1.76 (m, 2H) 1.87-1.93 (m, 2H) 2.00-2.07 (m, 2H)2.48-2.53 (m, 2H) 2.67-2.72 (m, 4H) 3.44-3.47 (m, 2H) 3.50-3.55 (m, 4H)7.24 (s, 1H) 7.61 (d, J=9.37 Hz, 2H) 7.86 (d, J=2.63 Hz, 1H) 8.09 (d,J=12.88 Hz, 1H) 8.48 (s, 1H) 9.06 (s, 1H) 11.41 (br s., 1H). LCMS (ESI)436 (M+H).

Example 104 Synthesis of Compound 104

Compound 104 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.29 (d, J=6.73 Hz, 6H) 1.66-1.79 (m, 2H) 1.84-1.95 (m,2H) 1.98-2.09 (m, 2H) 2.46-2.55 (m, 2H) 3.29-3.39 (m, 2H) 3.58-3.70 (m,4H) 3.77-3.86 (m, 4H) 7.24 (s, 1H) 7.66 (d, J=9.37 Hz, 1H) 7.96 (d,J=2.93 Hz, 1H) 8.08 (s, 1H) 8.48 (s, 1H) 9.06 (s, 1H) 9.28 (s, 1H) 9.67(s, 1H) 11.36 (s, 1H). LCMS (ESI) 447 (M+H).

Example 105 Synthesis of Compound 105

Compound 105 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.73 (s, 2H) 1.76-1.85 (m, 2H) 1.85-1.94 (m, 2H)1.98-2.07 (m, 2H) 2.19-2.26 (m, 2H) 2.48-2.52 (m, 1H) 2.70-2.81 (m, 4H)3.13-3.20 (m, 1H) 3.30-3.48 (m, 3H) 3.58-3.71 (m, 4H) 3.78-3.84 (m, 4H)7.24 (s, 1H) 7.62 (d, J=9.37 Hz, 2H) 7.89 (d, J=1.17 Hz, 1H) 8.09-8.18(m, 1H) 8.48 (s, 1H) 9.06 (s, 1H) 11.46 (br s., 1H). LCMS (ESI) 519(M+H).

Example 106 Synthesis of Compound 106

Compound 106 was synthesized using similar conditions to those describedfor compound 78 followed by the deblocking step described for compound65 and was converted to an HCl salt. ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.65-1.75 (m, 2H) 1.85-1.93 (m, 2H) 1.93-1.99 (m, 1H) 2.00-2.06 (m, 2H)2.08-2.14 (m, 1H) 2.47-2.55 (m, 2H) 3.07-3.25 (m, 2H) 3.25-3.69 (m, 5H)4.46 (s, 1H) 4.67 (s, 1H) 7.22 (s, 1H) 7.58-7.69 (m, 2H) 8.46 (s, 1H)9.02 (s, 1H) 9.34 (s, 1H) 9.65 (s, 1H). LCMS (ESI) 431 (M+H).

Example 107 Synthesis of Compound 107 (also referred to as Compound YY)

Compound 107 was synthesized using similar conditions to those describedfor compound 78 and was converted to an HCl salt. ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.65-1.82 (m, 3H) 1.89 (br. s., 2H) 1.98-2.08 (m, 2H)2.13 (br. s., 2H) 2.47-2.55 (m, 2H) 2.68 (d, J=4.98 Hz, 6H) 2.71-2.80(m, 2H) 3.29-3.71 (m, 10H) 7.16-7.26 (m, 1H) 7.67 (d, J=9.66 Hz, 2H)7.91 (d, J=2.05 Hz, 1H) 8.14 (br. s., 1H) 8.48 (br. s., 1H) 9.05 (s, 1H)11.14 (br. s., 1H) 11.43 (br. s., 1H). LCMS (ESI) 461 (M+H).

Example 108 Synthesis of Compound 108

Compound 108 was synthesized in a manner similar to that described forcompounds 64 and 65 and was recovered as an HCl salt. The analyticaldata was consistent with that described for the antipode compound 75.

Example 109 Synthesis of Compound 109

Compound 109 was synthesized in a manner similar to that described forcompounds 64 and 65 and was recovered as an HCl salt. The analyticaldata was consistent with that described for the antipode compound 75.

Example 110 Synthesis of Compound 110

Compound 110 was synthesized in a similar manner to that described forcompound 78 and then converted to its hydrochloride salt. ¹HNMR (600MHz, DMSO-d₆) δ ppm 1.50-1.65 (m, 1H) 1.92-2.02 (m, 3H) 2.06-2.15 (m,1H) 2.78 (d, J=3.81 Hz, 4H) 3.10-3.20 (m, 4H) 3.47-3.51 (m, 2H)3.64-3.71 (m, 1H) 3.76-3.83 (m, 2H) 3.98-4.14 (m, 1H) 7.20 (s, 2H) 7.77(s, 1H) 7.97 (s, 2H) 8.81 (s, 1H) 9.03 (s, 1H) 10.97 (br s., 1H). LCMS(ESI) 419 (M+H).

Example 111 Synthesis of Compound 111

Compound 111 was synthesized in a similar manner to that described forcompound 78 and then converted to its hydrochloride salt. ¹HNMR (600MHz, DMSO-d₆) δ ppm 1.54-1.59 (m, 1H) 1.92-2.01 (m, 3H) 2.06-2.15 (m,1H) 2.76-2.84 (m, 1H) 3.17-3.24 (m, 6H) 3.64-3.71 (m, 2H) 4.02-4.11 (m,2H) 7.22 (s, 2H) 7.64 (s, 1H) 7.97 (s, 2H) 8.75 (s, 1H) 8.97 (s, 1H)9.21 (s, 1H). LCMS (ESI) 405 (M+H).

Example 112 Synthesis of Compound 112

Compound 112 was synthesized using similar experimental conditions tothat described for compound 64.

Example 113 Synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate, Compound113

To a solution of 5-bromo-2,4-dichloropyrimidine (12.80 g, 0.054 mole) inethanol (250 mL) was added Hunig's base (12.0 mL) followed by theaddition of a solution of N-(tert-butoxycarbonyl)-1,2-diaminoethane (10g, 0.0624 mole) in ethanol (80 mL). The contents were stirred overnightfor 20 hrs. The solvent was evaporated under vacuum. Ethyl acetate (800mL) and water (300 mL) were added and the layers separated. The organiclayer was dried with magnesium sulfate and then concentrated undervacuum. Column chromatography on silica gel using hexane/ethyl acetate(0-60%) afforded tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)351 (M+H).

Example 114 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate, Compound 114

To tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate (5 g, 14.23mmole) in toluene (42 mL) and triethylamine (8.33 mL) under nitrogen wasadded triphenyl arsine (4.39 g), 3,3-diethoxyprop-1-yne (3.24 mL) andPddba (1.27 g). The contents were heated at 70 degrees for 24 hrs. Afterfiltration through CELITE®, the crude reaction was columned usinghexane/ethyl acetate (0-20%) to afford the desired product 3.9 g).Column chromatography of the resulting residue using hexane/ethylacetate (0-30%) afforded tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamate.LCMS (ESI) 399 (M+H).

Example 115 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 115

To a solution of Compound 114 (3.9 g, 0.00976 mole) in THF (60 mL) wasadded TBAF (68.3 mL, 7 eq). The contents were heated to 45 degrees for 2hrs. Concentration followed by column chromatography using ethylacetate/hexane (0-50%) afforded tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamateas a pale brown liquid (1.1 g). ¹HNMR (d₆-DMSO) δ ppm 8.88 (s, 1H), 6.95(brs, 1H), 6.69 (s, 1H), 5.79 (s, 1H), 4.29 (m, 2H), 3.59 (m, 4H), 3.34(m, 1H), 3.18 (m, 1H), 1.19 (m, 9H), 1.17 (m, 6H). LCMS (ESI) 399 (M+H).

Example 116 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-iodo-pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 116

To tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.1 g, 0.00025 mol) in acetonitrile (2 mL) was added1,3-diiodo-5,5-dimethylhydantoin (95 mg, 1 eq), and solid NaHCO₃ (63 mg,3 eq). The reacation was stirred at room tempreature for 16 hrs. Thereaction was filtered and concentrated in vacuo. The product waspurified by silica gel column chromatography using hexane/ethylacetate(0-50%) to afford tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-iodo-pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamateas a pale yellow solid (0.03 g). LCMS (ESI) 525 (M+H).

Example 117 Synthesis of tert-ButylN-[2-[2-chloro-6-(diethoxymethyl)-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate,Compound 117

To tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-iodo-pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.1 g, 0.19 mmole) in dioxane (3 mL) was added 2-methylphenylboronicacid (28 mg), tetrakis(triphenylphosphine)palladium (25 mg) andpotassium phosphate (250 mg) in water (0.3 mL). The reaction was heatedin a CEM Discovery microwave at 90° C. for 3 hrs. The crude reaction wasloaded onto silica gel and columned using hexane/ethyl acetate (0-30%)to afford tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.06 g). LCMS (ESI) 489 (M+H).

Example 118 Synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 118

To tert-butylN-[2-[2-chloro-6-(diethoxymethyl)-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate(0.85 g, 1.74 mmole) in AcOH (10 mL) was added water (1.5 mL). Thereaction was stirred at room temperature for 16 hrs. The crude reactionwas then concentrated under vacuum. After the addition of ethyl acetate(50 mL), the organic layer was washed with satd. NaHCO₃. The organiclayer was dried with magnesium sulfate and then concentrated undervacuum to afford the crude intermediate, tert-butylN-[2-[2-chloro-6-formyl-5-(o-tolyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate.To this crude intermediate in DMF (5 mL) was added oxone (1.3 g). Afterstirring for 2.5 hrs, water (20 mL) and ethyl acetate (100 mL) wereadded. The organic layer was separated, dried and then concentratedunder vacuum to afford the crude product which was columned over silicagel using hexane/ethyl acetate (0-50%) to afford7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.112 g). LCMS (ESI) 431 (M+H).

Example 119 Synthesis of Compound 119

To7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid (0.1 g, 0.261 mmol) in DCM (4.1 mL) was added DMAP (20 mg) followedby the addition of N,N′-diisopropylcarbodiimide (0.081 mL, 2 eq). Afterstirring for 3 hrs, TFA (0.723 mL) was added. Stirring was thencontinued for another 30 minutes. The reaction mixture was neutralizedwith satd. NaHCO₃. DCM (20 mL) was then added and the organic layerseparated, dried with magnesium sulfate and then concentrated undervacuum to afford the crude product which was columned usinghexane/ethylacetate (0-100%) to afford chloro tricyclic amide Compound119 (0.65 g). LCMS (ESI) 313 (M+H).

Example 120 Synthesis of Compound 120

To the chloro tricyclic amide (0.040 g, 0.128 mmole) (Compound 119) indioxane (2.5 mL) under nitrogen was added Pd₂(dba)₃ (12 mg), sodiumtert-butoxide (16 mg), BINAP (16 mg) and 4-morpholinoaniline (22.7 mg, 1eq). The reaction mixture was heated at 90° C. in a CEM Discoverymicrowave for 3.0 hrs. The crude reaction was loaded onto a silica gelcolumn and the contents eluted with DCM/MeOH (0-6%) to afford theproduct (10 mg). LCMS (ESI) 455 (M+H). ¹HNMR (600 MHz, DMSO-d₆) δ ppm2.14 (s, 3H) 3.23-3.50 (m, 2H) 3.57-3.73 (m, 2H), 3.81-3.92 (m, 8H),7.11-7.31 (m, 4H) 7.31-7.48 (m, 1H) 7.58-7.73 (m, 1H) 7.77-7.95 (m, 2H)8.05-8.21 (m, 1H) 8.44 (s, 1H) 9.85-10.01 (m, 1H).

Example 121 Synthesis of Compound 121

To the chloro tricyclic amide (0.024 g) (Compound 119) inN-methyl-2-pyrrolidone (NMP) (1.5 mL) was addedtrans-4-aminocyclohexanol (0.0768 mmol, 26.54 mg, 3 eq) and Hunig's base(0.4 mL). The reaction was heated in a CEM Discovery microwave vessel at150° C. for 1.2 hrs. The crude reaction was loaded onto a silica gelcolumn and the contents eluted with DCM/MeOH (0-10%) to afford theproduct (21 mg). LCMS (ESI) 392 (M+H). ¹HNMR (600 MHz, DMSO-d₆) δ ppm1.23 (d, J=8.78 Hz, 4H) 1.84 (br. s., 4H) 2.11 (s, 3H) 3.34-3.43 (m, 1H)3.55 (br. s., 2H) 3.72 (br. s., 1H) 4.13 (br. s., 2H) 4.50 (br. s., 1H)7.03 (br. s., 1H) 7.12-7.28 (m, 4H) 7.96 (br. s., 1H) 8.18 (br. s., 1H).

Example 122 Synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 122

7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 341 (M+H).

Example 123 Synthesis of Compound 123

Chloro tricyclic amide, Compound 123, was synthesized using a similarexperimental procedure as that described for the synthesis of chlorotricyclic amide (Compound 119). LCMS (ESI) 223 (M+H).

Example 124 Synthesis of Compound 124

To the chloro tricyclic amide, Compound 123 (0.035 g, 0.00157 mole) inNMP (1.5 mL) was added Hunig's base (0.3 mL) followed by the addition ofthe trans-4-aminocyclohexanol (54.2 mg). The reaction mixture was heatedat 150° C. for 1.5 hrs. The crude reaction was loaded onto a silica gelcolumn and the column was eluted with DCM/MeOH (0-10%) to afford theproduct (5 mg). LCMS (ESI) 302 (M+H).

Example 125 Synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamate,Compound 125

tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine withtert-butyl N-(2-amino-2-methyl-propyl)carbamate using similarexperimental conditions as described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)(M+H) 379.

Example 126 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamate,Compound 126

tert-butyl N-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamate was synthesized by treating tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewith 3,3-diethoxyprop-1-yne in the presence of a catalyst such as Pddbausing similar experimental conditions as described for the synthesis oftert-butyl N-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4 yl]amino] ethyl]carbamate LCMS (ESI) (M+H) 427.

Example 127 Synthesis of tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-2-methyl-propyl]carbamate,Compound 127

tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]-2-methyl-propyl]carbamatewas synthesized by treating tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butylN-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]ethyl]carbamate.LCMS (ESI) (M+H) 427.

Example 128 Synthesis of7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 128

7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 369 (M+H).

Example 129 Synthesis of Compound 129

Chloro tricyclic amide, Compound 129, was synthesized using a similarprocedure as that described for the synthesis of chloro tricyclic amide,Compound 119. LCMS (ESI) 251 (M+H).

Example 130 Synthesis of Compound 130

Compound 130 was synthesized by treating chlorotricyclic amine Compound129 with trans-4-aminocyclohexanol using similar experimental conditionsas for compound 124. LCMS (ESI) 330 (M+H). ¹HNMR (600 MHz, DMSO-d₆) δppm 1.07-1.34 (m, 4H) 1.47-2.05 (m, 10H) 3.09 (m, 1H) 3.51 (d, J=2.91Hz, 2H) 3.57 (m, 1H) 4.50 (br. s., 1H) 6.89 (s, 1H) 6.94-7.05 (m, 1H)8.04 (br. s., 1H) 8.60 (s, 1H) 9.00 (br. s., 1H).

Example 131 Synthesis of benzylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]propyl]carbamate,Compound 131

BenzylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]propyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine with benzylN-[1-(aminomethyl)propyl]carbamate using similar experimental conditionsas described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)(M+H) 413.

Example 132 Synthesis of benzylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]propyl]carbamate,Compound 132

BenzylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]propyl]carbamatewas prepared by treating benzylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]propyl]-carbamatewith 3,3-diethoxyprop-1-yne in the presence of a catalyst such as Pddbausing similar experimental conditions as described for the synthesis oftert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]ethyl]carbamateLCMS (ESI) (M+H) 461.

Example 133 Synthesis of benzylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]propyl]carbamate,Compound 133

BenzylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]propyl]carbamatewas synthesized by treating benzylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butyl N-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3d]pyrimidin-7-yl]ethyl]carbamate. LCMS (ESI) (M+H) 461.

Example 134 Synthesis of7-[2-(benzyloxycarbonylamino)butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 134

7-[2-(benzyloxycarbonylamino)butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 403 (M+H).

Example 135 Synthesis of Compound 135

To a solution of7-[2-(benzyloxycarbonylamino)butyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid in dichloromethane was added HBr, the reaction was stirred at 45degrees for 3 hrs. After concentration, 2N NaOH was added to basify(pH=8.0) the reaction followed by the addition of THF (20 mL). Boc₂O wasthen added (1.2 eq) and the reaction was stirred for 16 hrs. To thecrude reaction mixture was then added ethyl acetate (100 mL) and water(50 mL) and the organic phase was separated, dried (magnesium sulfate)and then concentrated under vacuum. To the crude product was addeddichloromethane (30 mL) followed by DIC and DMAP. After stirring for 2hrs, TFA was added and the contents stirred for an hour. The solventswere evaporated under vacuum and the residue basified with satd. NaHCO₃.Ethyl acetate was then added and the organic layer separated, dried(magnesium sulfate) and then concentrated under vacuum. Columnchromatography with hexane/ethyl acetate (0-100%) afforded the desiredchlorotricyclic core, Compound 135. LCMS (ESI) 251 (M+H).

Example 136 Synthesis of Compound 136

Compound 136 was synthesized by treating chlorotricyclic amine, Compound135, with trans-4-aminocyclohexanol using similar experimentalconditions as for compound 124. LCMS (ESI) 330 (M+H). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 0.80-0.95 (m, 3H) 1.35-1.92 (m, 10H) 3.66 (br. m., 3H)4.17 (br. s., 2H) 4.47 (br. s., 1H) 6.85 (s, 1H) 6.96 (br. s., 1H) 8.15(br. s., 1H) 8.62 (br. s., 1H).

Example 137 Synthesis of tert-butylN-[1[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]cyclopentyl]carbamate,Compound 137

tert-butylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]cyclopentyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine withtert-butyl N-[1-(aminomethyl)cyclopentyl]carbamate using similarexperimental conditions as described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)405 (M+H).

Example 138 Synthesis of tert-butylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]cyclopentyl]carbamate,Compound 138

tert-butylN-[1-[[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]methyl]cyclopentyl]carbamatewas synthesized by treating tert-butylN-[1-[[(5-bromo-2-chloro-pyrimidin-4-yl)amino]methyl]cyclopentyl]carbamatewith with 3,3-diethoxyprop-1-yne in the presence of a catalyst such asPddba using similar experimental conditions as described for thesynthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate LCMS (ESI) 453 (M+H).

Example 139 Synthesis of tert-butylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]cyclopentyl]carbamate,Compound 139

tert-butylN-[1-[[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]methyl]cyclopentyl]carbamatewas synthesized by treating tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butyl N-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3d]pyrimidin-7-yl]ethyl]carbamate. LCMS (ESI) 453 (M+H).

Example 140 Synthesis of7-[[1-(tert-butoxycarbonylamino)cyclopentyl]methyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid, Compound 140

7-[[1-(tert-butoxycarbonylamino)cyclopentyl]methyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 395 (M+H).

Example 141 Synthesis of Compound 141

Chlorotricyclic core Compound 141 was synthesized using a similarexperimental procedure as that described for the synthesis of chlorotricyclic amide Compound 119. LCMS (ESI) 277 (M+H).

Example 142 Synthesis of Compound 142

Compound 142 was synthesized by treating chlorotricyclic amine, Compound141, with trans-4-aminocyclohexanol using similar experimentalconditions as for Compound 124. LCMS (ESI) 356 (M+H). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.08-1.32 (m, 8H) 1.60-2.09 (m, 8H) 3.03-3.17 (m, 1H)3.35 (s, 2H) 3.54-3.62 (m, 1H) 4.51 (d, J=4.39 Hz, 1H) 6.88 (s, 1H) 6.96(br. s., 1H) 8.07 (br. s., 1H) 8.58 (s, 1H).

Example 143 Synthesis of tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamate,Compound 143

tert-butylN-[[1-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]cyclopentyl]methyl]carbamatewas synthesized by treating 5-bromo-2,4-dichloropyrimidine withtert-butyl N-[(1-aminocyclopentyl)methyl]carbamate using similarexperimental conditions as described for the synthesis of tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]ethyl]carbamate. LCMS (ESI)405 (M+H).

Example 144 Synthesis of tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamate,Compound 144

tert-butylN-[[1-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]cyclopentyl]methyl]carbamatewas synthesized by treating tert-butylN-[2-[(5-bromo-2-chloro-pyrimidin-4-yl)amino]-2-methyl-propyl]carbamatewith 3,3-diethoxyprop-1-yne in the presence of a catalyst such as Pddbausing similar experimental conditions as described for the synthesis oftert-butyl N-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4yl]amino]ethyl]carbamate.

LCMS (ESI) 453 (M+H).

Example 145 Synthesis of tert-butylN-[[1-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]cyclopentyl]methyl]carbamate,Compound 145

tert-ButylN-[[1-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3-d]pyrimidin-7-yl]cyclopentyl]methyl]carbamatewas synthesized by treating tert-butylN-[2-[[2-chloro-5-(3,3-diethoxyprop-1-ynyl)pyrimidin-4-yl]amino]-2-methyl-propyl]carbamatewith TBAF using similar experimental conditions as described for thesynthesis tert-butyl N-[2-[2-chloro-6-(diethoxymethyl)pyrrolo[2,3d]pyrimidin-7-yl]ethyl]carbamate. LCMS (ESI) 4534 (M+H).

Example 146 Synthesis of7-[2-(tert-butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6carboxylicacid, Compound 146

7-[2-(tert-Butoxycarbonylamino)-1,1-dimethyl-ethyl]-2-chloro-pyrrolo[2,3-d]pyrimidine-6-carboxylicacid was synthesized using a similar experimental procedure as thatdescribed for the synthesis of7-[2-(tert-butoxycarbonylamino)ethyl]-2-chloro-5-(o-tolyl)pyrrolo[2,3-d]pyrimidine-6-carboxylicacid. LCMS (ESI) 395 (M+H).

Example 147 Synthesis of Compound 147

Chloro tricyclic amide, Compound 147 was synthesized using a similarexperimental procedure as that described for the chloro tricyclic amide,Compound 119. LCMS (ESI) 277 (M+H).

Example 148 Synthesis of Compound 148

Compound 148 was synthesized by treating chlorotricyclic amine, Compound147, with trans-4-aminocyclohexanol using similar experimentalconditions as for Compound 124. LCMS (ESI) 356 (M+H). ¹HNMR (600 MHz,DMSO-d₆) δ ppm 1.06-1.35 (m, 8H) 1.45-1.95 (m, 8H) 3.10 (m, 1H) 3.58(br. s., 2H) 3.95 (br. s., 1H) 4.49 (br. s., 1H) 6.84 (s, 1H) 6.85-6.93(m, 1H) 8.29 (s, 1H) 8.61 (br. s., 1H).

Example 149 Synthesis of Compound 149

Step 1: Compound 59 is Boc protected according to the method of A.Sarkar et al. (JOC, 2011, 76, 7132-7140).

Step 2: Boc-protected Compound 59 is treated with 5 mol % NiCl₂(Ph₃)₂,0.1 eq triphenylphosphine, 3 eq Mn, 0.1 eq tetraethylammonium iodide, inDMI under CO₂ (1 atm) at 25° C. for 20 hours to convert the aryl halidederivative into the carboxylic acid.

Step 3: The carboxylic acid from Step 2 is converted to thecorresponding acid chloride using standard conditions.Step 4: The acid chloride from Step 3 is reacted with N-methylpiperazine to generate the corresponding amide.Step 5: The amide from Step 4 is deprotected using trifluoroacetic acidin methylene chloride to generate the target compound. Compound 149 ispurified by silica gel column chromatography eluting with adichloromethane—methanol gradient to provide Compound 149.

Each of Compounds 119 through 147 and corresponding compounds withvarious R⁸, R¹ and Z definitions may be reacted with sodium hydride andan alkyl halide or other halide to insert the desired R substitutionprior to reaction with an amine, such as described above for thesynthesis of Compound 120, to produce the desired product of Formulae I,II, III, IV, or V.

Example 150 CDK4/6 Inhibition In Vitro Assay

Selected compounds disclosed herein were tested in CDK4/cyclinD1,CDK2/CycA and CDK2/cyclinE kinase assays by Nanosyn (Santa Clara,Calif.) to determine their inhibitory effect on these CDKs. The assayswere performed using microfluidic kinase detection technology (CaliperAssay Platform). The compounds were tested in 12-point dose-responseformat in singlicate at Km for ATP. Phosphoacceptor substrate peptideconcentration used was 1 μM for all assays and Staurosporine was used asthe reference compound for all assays. Specifics of each assay are asdescribed below:

CDK2/CyclinA: Enzyme concentration: 0.2 nM; ATP concentration: 50 μM;Incubation time: 3 hr.

CDK2/CyclinE: Enzyme concentration: 0.28 nM; ATP concentration: 100 μM;Incubation time: 1 hr.

CDK4/CyclinD1: Enzyme concentration: 1 nM; ATP concentration: 200 μM;Incubation time: 10 hr.

The inhibitory IC₅₀ values for the compounds in Table 1 for CDK4/CycD1,CDK2/CycE, CDK2/CycA, as well as fold selectivity are presented in Table2.

TABLE 2 Selective Inhibition of CDK4 Fold Fold Selectivity SelectivityCDK4/ CDK2/ (CDK2/ CDK2/ (CDK2/ CycD1 IC₅₀ CycE IC₅₀ CycE/ CycA IC₅₀CycA/ Structure [nM] [nM] CDK4) [nM] CDK4) A 4.2 6350 1516 3160 754 B0.4 3040 6862 1890 4266 C 1.4 1920 1333 616 428 D 0.9 3480 3779 15001629 E 1 695 688 204 202 F 1.5 628 419 190 127 G 1.5 2580 1767 646 442 H1.5 1520 1013 377 251 I 2 2120 1065 1130 568 J 0.7 5110 7707 4340 6546 K1 1070 1019 738 703 L 5.7 4530 789 1490 260 M 2.3 2280 1004 1410 621 N 11500 1500 ND ND O 2.5 41410 1636 3150 1245 P 3.3 3560 1085 1010 308 Q0.6 1080 1722 3030 4833 R 0.5 1920 3918 1360 2776 S 1.7 1250 718 342 197T 0.8 1660 2022 1670 2034 U 0.7 1460 2229 857 1308 V 2.9 3500 1224 2130745 W 2.7 3970 1481 539 201 X 0.9 11600 12975 1840 2058 Y 2.5 124 50 6125 Z 3.2 3710 1174 647 205 AA 0.5 6100 13319 4630 10109 BB 0.8 1680 2017502 603 CC 1.6 1250 791 755 478 DD 1.9 9620 5200 8360 4519 EE 3.8 1660432 1110 289 FF 1.2 4620 3949 1400 1197 GG 1 3580 3377 1510 1425 HH 1.71280 766 265 159 II 2 367 184 239 120 JJ 1.4 288 204 ND ND KK 2.3 1760762 915 396 LL 2 202 103 108 55 MM 1.8 3390 1863 597 328 NN 3.7 47001274 1560 423 OO 9 3980 442 570 63 PP 3.1 3600 1146 3090 984 QQ 4.1 3060746 2570 627 RR 1.2 1580 1374 693 603 SS 0.8 1460 1865 1390 1775 TT 0.81260 1550 596 733 UU 7.3 3960 542 ND ND VV 3.3 2630 809 789 243 WW 0.71350 204 ND ND XX 1.3 7300 5615 6290 4838 YY 4.6 6900 1490 ND ND ZZ 10.59960 949 ND ND AAA 2.3 6010 2591 2130 918 BBB 2.8 187 68 85 31 CCC 22170 1074 457 226 DDD 9.5 9350 986 ND ND EEE 0.2 2950 1266 943 405 FFF4.7 4540 966 1370 291 GGG 13.7 7610 555 ND ND HHH 6.8 2840 419 ND ND III6 3770 626 ND ND JJJ 3.2 5200 1620 2830 882 KKK 1.3 291 231 87.3 69 LLL3.2 1620 509 4530 1425 MMM 3.2 1890 600 990 314 NNN 1.4 2930 2154 1010743 OOO 2.4 393 164 203 85 PPP 0.8 16500 21263 2640 3402 QQQ 10.5 111001057 ND ND RRR 2.6 4500 1758 ND ND SSS 2 2280 1112 1880 917 TTT 3.4 3030899 ND ND UUU 18 16460 914 ND ND VVV 7.4 4380 589 ND ND WWW 18.5 2500135 ND ND XXX 11.4 6620 581 ND ND

To further characterize its kinase activity, Compound T was screenedagainst 456 (395 non-mutant) kinases using DiscoveRx's KINOMEscan™profiling service. The compound was screened using a singleconcentration of 1000 nM (>1000 times the IC50 on CDK4). Results fromthis screen confirmed the high potency against CDK4 and high selectivityversus CDK2. Additionally, the kinome profiling showed that Compound Twas relatively selective for CDK4 and CDK6 compared to the other kinasestested. Specifically, when using an inhibitory threshold of 65%, 90%, or99%, Compound T inhibited 92 (23.3%), 31 (7.8%) or 6 (1.5%) of 395non-mutant kinases respectively.

In addition to CDK4 kinase activity, several compounds were also testedagainst CDK6 kinase activity. The results of the CDK6/CycD3 kinaseassays, along with the CDK4/cyclinD1, CDK2/CycA and CDK2/cyclinE kinaseassays, are shown for PD0332991 (Reference) and the compounds T, Q, GG,and U in Table 3. The IC₅₀ of 10 nM for CDK4/cyclinD1 and 10 uM forCDK12/CyclinE agrees well with previously published reports forPD0332991 (Fry et al. Molecular Cancer Therapeutics (2004)3(11)1427-1437; Toogood et al. Journal of Medicinal Chemistry (2005) 48,2388-2406). Compounds T, Q, GG, and U are more potent (lower IC₅₀) withrespect to the reference compound (PD0332991) and demonstrate a higherfold selectivity with respect to the reference compound (CDK2/CycE IC₅₀divided by CDK4/CycD1 IC₅₀).

TABLE 3 Inhibition of CDK kinases by Compounds T, Q, GG, and U FoldCDK4/ CDK2/ Selectivity CDK2/ CDK6/ CycD1 CycE CDK2/ CycA CycD3 FormulaIC₅₀(nM) IC₅₀(uM) CDK4 IC₅₀(uM) IC₅₀(nM) PD0332991 10 10 1000 Not NotReference determined determined Compound 0.821 1.66 2022 1.67 5.64 TCompound 0.627 1.08 1722 3.03 4.38 Q Compound 1.060 3.58 3377 1.51 4.70GG Compound 0.655 1.46 2229 .857 5.99 U

Example 151 G1 Arrest (Cellular G1 and S-Phase) Assay

For determination of cellular fractions in various stages of the cellcycle following various treatments, HS68 cells (human skin fibroblastcell line (Rb-positive)) were stained with propidium iodide stainingsolution and run on Dako Cyan Flow Cytometer. The fraction of cells inG0-G1 DNA cell cycle versus the fraction in S-phase DNA cell cycle wasdetermined using FlowJo 7.2 0.2 analysis.

The compounds listed in Table 1 were tested for their ability to arrestHS68 cells at the G1 phase of the cell cycle. From the results of thecellular G1 arrest assay, the range of the inhibitory EC₅₀ valuesnecessary for G1 arrest of HS68 cells was from 22 nM to 1500 nM (seecolumn titled “Cellular G1 Arrest EC₅₀” in Table 4).

Example 152 Inhibition of Cellular Proliferation

Cellular proliferation assays were conducted using the following cancercell lines: MCF7 (breast adenocarcinoma—Rb-positive), ZR-75-1 (breastductal carcinoma—Rb-positive), H69 (human small cell lungcancer—Rb-negative) cells, or A2058 (human metastatic melanoma cells—Rb-negative). These cells were seeded in Costar (Tewksbury, Mass.) 309396 well tissue culture treated white walled/clear bottom plates. Cellswere treated with the compounds of Table 1 as nine point dose responsedilution series from 10 uM to 1 nM. Cells were exposed to compounds andthen cell viability was determined after either four (H69) or six (MCF7,ZR75-1, A2058) days as indicated using the CellTiter-Glo® luminescentcell viability assay (CTG; Promega, Madison, Wis., United States ofAmerica) following the manufacturer's recommendations. Plates were readon BioTek (Winooski, Vt.) Syngergy2 multi-mode plate reader. TheRelative Light Units (RLU) were plotted as a result of variable molarconcentration and data was analyzed using Graphpad (LaJolla, Calif.)Prism 5 statistical software to determine the EC₅₀ for each compound.

The results of the cellular inhibition assays for the two Rb-positivebreast cancer cell lines (MCF7 and ZR75-1) are shown in Table 4. Therange of the inhibitory EC₅₀ values necessary for inhibition of MCF7breast cancer cell proliferation was 28 nM to 257 nM. The range of theinhibitory EC₅₀ values necessary for inhibition of ZR75-1 breast cancercell proliferation was 24 nM to 581 nM.

Examples of representative compounds highly active against theproliferation of MCF7 breast adenocarcinoma cells are shown in FIGS.21-24. The compounds tested in FIGS. 21-24 (Compounds T, Q, GG, U, H,MM, 00, and PD-332991) all showed significant inhibition of cellularproliferation of MCF-7 cells. As can be seen in FIG. 21, compound Tshows more potent activity against MCF-7 cells than PD0332991.

Examples of representative compounds highly active against theproliferation of ZR75-1 (breast ductal carcinoma (Rb-Positive)) cellsare shown in FIGS. 25-28. The compounds tested in FIGS. 25-28 (CompoundsT, Q, GG, U, H, MM, OO, and PD-332991) all showed significant inhibitionof cellular proliferation of ZR75-1 cells. As can be seen in FIG. 25,compound T shows more potent activity against ZR75-1 cells thanPD0332991.

In addition to breast cancer cell lines, a number of the compoundsdisclosed herein were also evaluated against a small cell lung cancercell line (H69) and a human metastatic melanoma cell line (A2058), twoRb-deficient (Rb-negative) cell lines. The results of these cellularinhibition assays are shown in Table 4. The range of the inhibitory EC₅₀values necessary for inhibition of H69 small cell lung cancer cells was2040 nM to >3000 nM. The range of the inhibitory EC₅₀ values necessaryfor inhibition of A2058 malignant melanoma cell proliferation was 1313nM to >3000 nM. In contrast to the significant inhibition seen on thetwo Rb-positive breast cancer cell lines, it was found that thecompounds tested were not significantly effective at inhibitingproliferation of the small cell lung cancer or melanoma cells.

TABLE 4 Inhibition of Cancer Cell Proliferation Cellular G1 MCF7 ZR75-1H69 A2058 Arrest Cellular Cellular Cellular Cellular EC₅₀ EC₅₀ EC₅₀ EC₅₀EC₅₀ Structure (nM) [nM] [nM] [nM] [nM] A 110 75 44 >3000 ND B 90 201245 ND ND C 95 88 73 ND ND D 50 57 46 2911 1670 E 75 53 62 2580 1371 F175 ND ND ND ND G 175 ND ND ND ND H 85 85 120 2040 1313 I 80 61 40 29501062 J 110 70 82 >3000 >3000 K 28 43 ND >3000 1787 L 65 506 ND2161 >3000 M 100 ND ND ND ND N 25 28 24 >3000 1444 O 40 56 29 >3000 2668P 30 60 43 >3000 >3000 Q 100 49 35 >3000 2610 R 70 36 50 >3000 2632 S150 76 ND >3000 >3000 T 100 49 36 >3000 >3000 U 25 70 59 >3000 >3000 V70 50 29 >3000 1353 W 160 294 ND >3000 >3000 X 65 ND ND >3000 >3000 Y350 ND ND ND ND Z 110 141 54 ND ND AA 70 47 47 >3000 ND BB 75 ND ND 29431635 CC 90 50 38 >3000 >3000 DD 100 ND ND ND ND EE 125 216 203 ND ND FF80 140 ND ND ND GG 80 52 62 2920 2691 HH 110 ND ND ND ND II 40 9433 >3000 >3000 JJ 90 122 ND >3000 >3000 KK 22 333 ND 2421 1379 LL 125 96ND >3000 >3000 MM 100 73 77 >3000 >3000 NN 110 ND ND ND ND OO 95 120229 >3000 >3000 PP 100 164 66 ND ND QQ 120 ND ND >3000 >3000 RR 90 72 ND2888 1617 SS 80 94 53 2948 1658 TT 75 ND ND ND ND UU 300 ND ND ND ND VV200 ND ND ND ND WW 400 ND ND ND ND XX 225 ND ND ND ND YY 175 257 581 NDND ZZ 500 ND ND ND ND AAA 275 320 ND >3000 >3000 BBB 230 123ND >3000 >3000 CCC 250 ND ND ND ND DDD 350 ND ND ND ND EEE 250 453ND >3000 >3000 FFF 650 ND ND ND ND GGG 350 ND ND ND ND HHH 250 ND ND NDND III 250 ND ND ND ND JJJ 240 ND ND ND ND KKK 190 ND ND ND ND LLL 250ND ND ND ND MMM 200 134 141 >3000 >3000 NNN 210 ND ND ND ND OOO 200 138ND >3000 >3000 PPP 275 ND ND ND ND QQQ 500 ND ND ND ND RRR 400 ND ND NDND SSS 1500 ND ND ND ND TTT 350 ND ND ND ND UUU 300 ND ND ND ND VVV 300ND ND ND ND WWW 300 ND ND ND ND XXX 300 ND ND ND ND

Example 153 HSPC Growth Suppression Studies

The effect of PD0332991 on HSPCs has been previously demonstrated. FIG.2 shows the EdU incorporation of mice HSPC and myeloid progenitor cellsfollowing a single dose of 150 mg/kg PD0332991 by oral gavage to assessthe temporal effect of transient CDK4/6 inhibition on bone marrow arrestas reported in Roberts et al. Multiple Roles of Cyclin-Dependent Kinase4/6 Inhibitors in Cancer Therapy. JCNI 2012; 104(6):476-487 (FIG. 2A).As can be seen in FIG. 2, a single oral dose of PD0332991 results in asustained reduction in HSPC (LKS+) and myeloid progenitor cells (LKS−)for greater than 36 hours. Not until 48 hours post oral dosing do HSPCand myeloid progenitor cells return to baseline cell division.

Example 154 Pharmacokinetic and Pharmacodynamic Properties ofAnti-Neoplastic Compounds

Compounds of the present invention demonstrate good pharmacokinetic andpharmacodynamic properties. Compound T, Q, GG, and U were dosed to miceat 30 mg/kg by oral gavage or 10 mg/kg by intravenous injection. Bloodsamples were taken at 0, 0.25, 0.5, 1.0, 2.0, 4.0, and 8.0 hours postdosing and the plasma concentration of compound T, Q, GG, or U weredetermined by HPLC. Compound T, GG, and U were demonstrated to haveexcellent oral pharmacokinetic and pharmacodynamic properties as shownin Table 5. This includes very high oral bioavailability (F (%)) of 52%to 80% and a plasma half-life of 3 to 5 hours following oraladministration. Compound T, Q, GG, and U were demonstrated to haveexcellent pharmacokinetic and pharmacodynamic properties when deliveredby intravenous administration. Representative IV and oral PK curves forall four compounds are shown in FIG. 3.

TABLE 5 Pharmacokinetic and pharmacodynamic properties ofanti-neoplastic compounds Compound Compound Compound Compound Mouse PK TQ GG U CL (mL/min/kg) 35 44 82 52 Vss (L/kg) 2.7 5.2 7.5 3.4 t_(1/2) (h)p.o. 5 0.8 3.5 3 AUC_(0-inf) (uM*h) i.v. 1.3 0.95 1.1 0.76 AUC (uM*h)p.o. 2.9 0.15 1.9 3.3 C_(max) (uM) p.o. 2.5 0.16 1.9 4.2 T_(max) (h)p.o. 1 0.5 1 0.5 F (%) 80 2 52 67

Example 155 Cellular Wash-Out Experiment

HS68 cells were seeded out at 40,000 cells/well in 60 mm dish on day 1in DMEM containing 10% fetal bovine serum, 100 U/mlpenicillin/streptomycin and 1× Glutamax (Invitrogen) as described(Brookes et al. EMBO J, 21(12)2936-2945 (2002) and Ruas et al. Mol CellBiol, 27(12)4273-4282 (2007)). 24 hrs post seeding, cells are treatedwith compound T, compound Q, compound GG, compound U, PD0332991, or DMSOvehicle alone at 300 nM final concentration of test compounds. On day 3,one set of treated cell samples were harvested in triplicate (0 Hoursample). Remaining cells were washed two times in PBS-CMF and returnedto culture media lacking test compound. Sets of samples were harvestedin triplicate at 24, 40, and 48 hours.

Alternatively, the same experiment was done using normal Renal ProximalTubule Epithelial Cells (Rb-positive) obtained from American TypeCulture Collection (ATCC, Manassas, Va.). Cells were grown in anincubator at 37° C. in a humidified atmosphere of 5% CO2 in RenalEpithelial Cell Basal Media (ATCC) supplemented with Renal EpithelialCell Growth Kit (ATCC) in 37° C. humidified incubator.

Upon harvesting cells, samples were stained with propidium iodidestaining solution and samples run on Dako Cyan Flow Cytometer. Thefraction of cells in G0-G1 DNA cell cycle versus the fraction in S-phaseDNA cell cycle was determined using FlowJo 7.2 0.2 analysis.

FIG. 4 shows cellular wash-out experiments which demonstrate theinhibitor compounds of the present invention have a short, transientG1-arresting effect in different cell types. Compounds T, Q, GG, and Uwere compared to PD0332991 in either human fibroblast cells(Rb-positive) (FIGS. 4A & 4B) or human renal proximal tubule epithelialcells (Rb-positive) (FIGS. 4C & 4D) and the effect on cell cyclefollowing washing out of the compounds was determined at 24, 36, 40, and48 hours.

As shown in FIG. 4 and similar to results in vivo as shown in FIG. 2,PD0332991 required greater than 48 hours post wash out for cells toreturn to normal baseline cell division. This is seen in FIG. 4A andFIG. 4B as values equivalent to those for the DMSO control for eitherthe G0-G1 fraction or the S-phase of cell division, respectively, wereobtained. In contrast, HS68 cells treated with compounds of the presentinvention returned to normal baseline cell division in as little as 24hours or 40 hours, distinct from PD0332991 at these same time points.The results using human renal proximal tubule epithelial cells (FIGS. 4C& 4D) also show that PD0332991-treated cells took significantly longerto return to baseline levels of cell division as compared to cellstreated with compounds T, Q, GG, or U.

Example 156 Bone Marrow Proliferation as Evaluated Using EdUIncorporation and Flow Cytometry Analysis

For HSPC proliferation experiments, young adult female FVB/N mice weretreated with a single dose as indicated of compound T, compound Q,compound GG or PD0332991 by oral gavage. Mice were then sacrificed atthe indicated times (0, 12, 24, 36, or 48 hours following compoundadministration), and bone marrow was harvested (n=3 mice per timepoint), as previously described (Johnson et al. J. Clin. Invest. (2010)120(7), 2528-2536). Four hours before the bone marrow was harvested,mice were treated with 100 μg of EdU by intraperitoneal injection(Invitrogen). Bone marrow mononuclear cells were harvested andimmunophenotyped using previously described methods and percent EdUpositive cells were then determined (Johnson et al. J. Clin. Invest.(2010) 120(7), 2528-2536). In brief, HSPCs were identified by expressionof lineage markers (Lin−), Sca1 (S+), and c-Kit (K+).

Analysis in mice determined that Compound T, Compound Q, and Compound GGdemonstrated dose dependent, transient, and reversible G1-arrest of bonemarrow stem cells (HSPC) (FIG. 5). Six mice per group were dosed by oralgavage at 150 mg/kg of Compound T, Compound Q, Compound GG, or vehicleonly. Four hours before animals were sacrificed and the bone marrow washarvested, mice were treated with 100 μg of EdU by intraperitonealinjection. Three mice per group were sacrificed at 12 hours and theremaining three animals per group were sacrificed at 24 hours. Resultsare shown in FIG. 5A as the ratio of EdU positive cells for treatedanimals at 12 or 24 hour time points compared to control. Compound T andGG demonstrated a reduction in EdU incorporation at 12 hours which wasstarting to return to normal at 24 hours. Compound Q also demonstratedsome reduction at 12 hours and started to return to baseline at 24 hoursdespite the fact that oral bioavailability of Compound Q is low.

Further experiments were completed with Compound T examining doseresponse and longer periods of compound treatment. Compound T was dosedby oral gavage at 50, 100 or 150 mg/kg and EdU incorporation into bonemarrow was determined at 12 and 24 hours as described above.Alternatively, Compound T was dosed by oral gavage at 150 mg/kg and EdUincorporation into bone marrow was determined at 12, 24, 36 and 48hours. As can be seen in FIGS. 5B and 5C, and similar to the cellularwashout experiments, bone marrow cells, and in particular HSPCs werereturning to normal cell division as determined by EdU incorporation in24 hours following oral gavage at a number of doses. The 150 mg/kg oraldose of Compound Tin FIG. 5C can be compared directly to the results ofthe same dose of PD0332991 shown in FIG. 2 where cells were stillnon-dividing (as determined by low EdU incorporation) at 24 and 36hours, only returning to normal values at 48 hours.

Example 157 HSPC Growth Suppression Studies Comparing Compound T andPD0332991

FIG. 6 is a graph of the percentage of EdU positive HSPC cells for micetreated with either PD0332991 (triangles) or compound T (upside downtriangles) v. time after administration (hours) of the compound. Bothcompounds were administered at 150 mg/kg by oral gavage. One hour priorto harvesting bone marrow, EdU was IP injected to label cycling cells.Bone marrow was harvested at 12, 24, 36, and 48 hours after compoundtreatment and the percentage of EdU positive HSPC cells was determinedat each time point.

As seen in FIG. 6, a single oral dose of PD0332991 results in asustained reduction in HSPCs for greater than 36 hours. In contrast, asingle oral dose of Compound T results in an initial reduction of HSPCproliferation at 12 hours, but proliferation of HSPCs resumes by 24hours after dosage of Compound T.

Example 158 Metabolic Stability

The metabolic stability of Compound T in comparison to PD0332991 wasdetermined in human, dog, rat, monkey, and mouse liver microsomes.Human, mouse, and dog liver microsomes were purchased from Xenotech, andSprague-Dawley rat liver microsomes were prepared by Absorption Systems.The reaction mixture comprising 0.5 mg/mL of liver microsomes, 100 mM ofpotassium phosphate, pH 7.4, 5 mM of magnesium chloride, and 1 uM oftest compound was prepared. The test compound was added into thereaction mixture at a final concentration of 1 uM. An aliquot of thereaction mixture (without cofactor) was incubated in shaking water bathat 37 deg. C. for 3 minutes. The control compound, testosterone, was runsimultaneously with the test compound in a separate reaction. Thereaction was initiated by the addition of cofactor (NADPH), and themixture was then incubated in a shaking water bath at 37 deg. C.Aliquots (100 μL) were withdrawn at 0, 10, 20, 30, and 60 minutes forthe test compound and 0, 10, 30, and 60 minutes for testosterone. Testcompound samples were immediately combined with 100 μL of ice-coldacetonitrile containing internal standard to terminate the reaction.Testosterone samples were immediately combined with 800 μL of ice cold50/50 acetonitrile/dH2O containing 0.1% formic acid and internalstandard to terminate the reaction. The samples were assayed using avalidated LC-MS/MS method. Test compound samples were analyzed using theOrbitrap high resolution mass spectrometer to quantify the disappearanceof parent test compound and detect the appearance of metabolites. Thepeak area response ration (PARR) to internal standard was compared tothe PARR at time 0 to determine the percent of test compound or positivecontrol remaining at time-point. Half-lives were calculated usingGraphPad software, fitting to a single-phase exponential decay equation.

Half-life was calculated based on t1/2=0.693k, where k is theelimination rate constant based on the slope plot of natural logarithmpercent remaining versus incubation time. When calculated half-life waslonger than the duration of the experiment, the half-life was expressedas >the longest incubation time. The calculated half-life is also listedin parentheses. If the calculated half-life is >2× the duration of theexperiment, no half-life was reported. The timely resumption of cellularproliferation is necessary for tissue repair, and therefore an overlylong period of arrest is undesirable in healthy cells such as HSPCs. Thecharacteristics of a CDK4/6 inhibitor that dictate its arrestingduration are its pharmacokinetic (PK) and enzymatic half-lives. Onceinitiated, a G1-arrest in vivo will be maintained as long as circulatingcompound remains at an inhibitory level, and as long as the compoundengages the enzyme. PD032991, for example, possesses an overall long PKhalf-life and a fairly slow enzymatic off-rate. In humans, PD0332991exhibits a PK half-life of 27 hours (see Schwartz, G K et al. (2011)BJC, 104:1862-1868). In humans, a single administration of PD0332991produces a cell cycle arrest of HSPC lasting approximately one week.This reflects the 6 days to clear the compound (5 half-lives×27 hourhalf-life), as well as an additional 1.5 to 2 days of inhibition ofenzymatic CDK4/6 function. This calculation suggests that it takes atotal of 7+days for normal bone marrow function to return, during whichtime new blood production is reduced. These observations may explain thesevere granulocytopenia seen with PD0332991 in the clinic.

Further experiments were completed with Compound T and PD0332991 tocompare the metabolic stability (half-life) in human, dog, rat, monkey,and mouse liver microsomes. As shown in FIG. 7, when analyzing thestability of the compounds in liver microsomes across species, thedeterminable half-life of Compound T is shorter in each species comparedto that reported for PD0332991. Furthermore, as previously describedabove and in FIG. 4, it appears that PD0332991 also has an extendedenzymatic half-life, as evidenced by the production of a pronounced cellcycle arrest in human cells lasting more than forty hours even aftercompound is removed from the cell culture media (i.e., in an in vitrowash-out experiment). As further shown in FIG. 4, removal of thecompounds described herein from the culture media leads to a rapidresumption of proliferation, consistent with a rapid enzymatic off rate.These differences in enzymatic off rates translate into a markeddifference in pharmacodynamic (PD) effect, as shown in FIGS. 2, 5C, and6. As shown, a single oral dose of PD0332991 produces a 36+hour growtharrest of hematopoietic stem and progenitor cells (HSPCs) in murine bonemarrow, which is greater than would be explained by the 6 hour PKhalf-life of PD0332991 in mice. In contrast, the effect of Compound T ismuch shorter, allowing a rapid re-entry into the cell cycle, providingexquisite in vivo control of HSPC proliferation.

Example 159 Efficacy of the CDK4/6 Inhibitor, Compound T) in HER2-DrivenBreast Tumors

A HER2-driven model (Rb-positive) of breast cancer (Muller W J, Sinn E,Pattengale P K, Wallace R, Leder P. Single-step induction of mammaryadenocarcinoma in transgenic mice bearing the activated c-neu oncogene.Cell 1988; 54: 105-15), that expresses c-neu (the mouse ortholog ofhuman HER2) driven by the MMTV promoter was used in the followingexample. This model was chosen because previous studies in murine (Yu Q,Geng Y, Sicinski P. Specific protection against breast cancers by cyclinD1 ablation. Nature 2001; 411: 1017-21; Landis M W, Pawlyk B S, Li T,Sicinski P, Hinds P W. Cyclin D1-dependent kinase activity in murinedevelopment and mammary tumorigenesis. Cancer Cell 2006; 9: 13-22; ReddyH K, Mettus R V, Rane S G, Grana X, Litvin J, Reddy E P.Cyclin-dependent kinase 4 expression is essential for neu-induced breasttumorigenesis. Cancer Res 2005; 65: 10174-8; Yu Q, Sicinska E, Geng Y,Ahnstrom M, Zagozdzon A, Kong Y, et al. Requirement for CDK4 kinasefunction in breast cancer. Cancer Cell 2006; 9: 23-32.) and humanHER2-positive breast cancer (An H X, Beckmann M W, Reifenberger G,Bender H G, Niederacher D. Gene amplification and overexpression of CDK4in sporadic breast carcinomas is associated with high tumor cellproliferation. Am J Pathol 1999; 154: 113-8; Samady L, Dennis J,Budhram-Mahadeo V, Latchman D S. Activation of CDK4 gene expression inhuman breast cancer cells by the Brn-3b POU family transcription factor.Cancer Biol Ther 2004; 3: 317-23; Takano Y, Takenaka H, Kato Y, MasudaM, Mikami T, Saegusa M, et al. Cyclin D1 overexpression in invasivebreast cancers: correlation with cyclin-dependent kinase 4 and oestrogenreceptor overexpression, and lack of correlation with mitotic activity.J Cancer Res Clin Oncol 1999; 125: 505-12) suggest that these tumorsrequire CDK4/6 and CCND1 for progression and maintenance.

MMTV-neu mice were generated and observed post-lactation, with tumorsobserved with a median latency of approximately 25 weeks. Mice wereenrolled in therapy studies when tumors reached a standard size (50-60mm3) that permitted easy serial assessment. Tumor-bearing mice werecontinuously treated with Compound T added to their chow (100 mg/kg/d or150 mg/kg/d). MMTV-c-neu (control, n=9; Compound T 100 mg/kg, n=7;Compound T 150 mg/kg, n=6) mice were examined weekly to assess tumordevelopment by palpation. Tumor volumes were calculated by the formula,Volume=[(width)²×length]/2. Tumor-bearing mice were euthanized at theindicated times due to predefined morbidity, tumor ulceration, or atumor size of more than 1.5 cm in diameter.

As shown in FIGS. 8 and 9, continuous treatment with Compound T (100mg/kg/d or 150 mg/kg/d) led to a marked reduction in tumor volume duringa 28 day course of therapy. Several tumors displayed complete tumorregression and no resistance to Compound T was noted during the 28 daycourse of therapy. These data show that this HER2-driven mouse model is‘addicted’ to CDK4/6 activity for proliferation and Compound T is aneffective agent in CDK4/6 dependent, Rb-positive tumors.

Example 160 Efficacy of CDK4/6 Inhibitors (Compound T, Compound GG, andCompound U) in HER2-Driven Breast Tumors

Preclinical characterization of compound T, compound GG, and compound Uindicates that they inhibit CDK4 and CDK6 with an IC₅₀ of 0.7-1.0 nM and5-6 nM, respectively. In tumor cells with functional Rb protein, thesecompounds potently inhibit Rb phosphorylation resulting in a G1 arrest.The in vivo efficacy of the CDK4/6 inhibitors compound T, compound GG,and compound U was tested in the genetically engineered mouse model ofluminal breast cancer. Tumors were serially assessed weekly usingcaliper measurements. Therapeutic intervention began once tumors reached40-64 mm³. Tumor volume was calculated using the formula((Width)×Length)/2. All three compounds were administered orally viamedicated diets (100 mg/kg/d). Medicated diets were administered for 28consecutive days and then stopped. RECIST criteria were used to assessobjective response rates. The objective response rates were categorizedbased on the percentage change in tumor volume, using the followingcategories: CR (complete response)=100% response; PR (partialresponse)=at least a 30% decrease; SD (stable disease)=no change (not aPR and not a PD); and PD (progressive disease)=20% increase.

As shown in FIG. 10, objective responses were noted in mice treated withcompound T, compound GG, and compound U. Treatment groups were welltolerated with no clinical signs of toxicity, no weight loss and nodeaths associated with toxicity. The linear regression t-test was usedto determine statistical significance of tumor volume growth over time.All three cohorts were statistically significant compared to non-treatedcohorts; compound T, p<0.0001; compound GG, p=0.0001; compound U,p<0.0001. As shown in FIG. 10, the number of animals in each objectivecategory was determined. Compound T was found to have a 100% objectiveresponse rate (n=7), Compound GG was found to have an 85% objectiveresponse rate (n=7), and Compound U was found to have a 100% objectiveresponse rate (n=8). In FIG. 11, the tumor volumes from the MMTV-Neumice treated with compound T, compound GG, and compound U are shown,with tumor volumes being measured every seven days. In FIG. 12, the datafor the best response (14 days or later) is shown for each individualtumor. Taken together, these data show that continuous treatment withcompound T (100 mg/kg), compound GG, (100 mg/kg), or compound U (100mg/kg) led to a marked decrease in tumor volume during a 28 day courseof therapy.

Example 161 Cell Cycle Arrest by Compound T in CDK4/6-Dependent Cells

To test the ability of CDK4/6 inhibitors to induce a clean G1-arrest, acell based screening method was used consisting of two CDK4/6-dependentcell lines (tHS68 and WM2664; Rb-positive) and one CDK4/6-independent(A2058; Rb-negative) cell line. Twenty-four hours after plating, eachcell line was treated with Compound T in a dose dependent manner for 24hours. At the conclusion of the experiment, cells were harvested, fixed,and stained with propidium iodide (a DNA intercalator), which fluorescesstrongly red (emission maximum 637 nm) when excited by 488 nm light.Samples were run on Dako Cyan flow cytometer and >10,000 events werecollected for each sample. Data were analyzed using FlowJo 2.2 softwaredeveloped by TreeStar, Inc.

In FIG. 29A, results show that Compound T induces a robust G1 cell cyclearrest, as nearly all cells are found in the G0-G1 phase upon treatmentwith increasing amounts of Compound T. In FIG. 29A, the results showthat in CDK4/6-dependent cell lines, Compound T induced a robust G1 cellcycle arrest with an EC₅₀ of 80 nM in tHS68 cells with a correspondingreduction in S-phase ranging from 28% at baseline to 6% at the highestconcentration shown. Upon treatment with Compound T (300 nM), there wasa similar reduction in the S-phase population and an increase inG1-arrested cells in both CDK4/6-dependent cell lines (tHS68 (CompareFIGS. 29B and 29E) and WM2664 (Compare FIGS. 29C and 29F)), but not inthe CDK4/6-independent (A2058; Compare FIGS. 29D and 29G) cell line. TheCDK4/6-independent cell line shows no effect in the presence ofinhibitor.

Example 162 Compound T Inhibits Phosphorylation of RB

The CDK4/6-cyclin D complex is essential for progression from G1 to theS-phase of the DNA cell cycle. This complex phosphorylates theretinoblastoma tumor suppressor protein (Rb). To demonstrate the impactof CDK4/6 inhibition on Rb phosphorylation (pRb), Compound T was exposedto three cell lines, two CDK4/6 dependent (tHS68, WM2664; Rb-positive)and one CDK4/6 independent (A2058; Rb-negative). Twenty four hours afterseeding, cells were treated with Compound T at 300 nM finalconcentration for 4, 8, 16, and 24 hours. Samples were lysed and proteinwas assayed by western blot analysis. Rb phosphorylation was measured attwo sites targeted by the CDK4/6-cyclin D complex, Ser780 and Ser807/811using species specific antibodies. Results demonstrate that Compound Tblocks Rb phosphorylation in Rb-dependent cell lines by 16 hours postexposure, while having no effect on Rb-independent cells (FIG. 30).

This specification has been described with reference to embodiments ofthe invention. The invention has been described with reference toassorted embodiments, which are illustrated by the accompanyingExamples. The invention can, however, be embodied in different forms andshould not be construed as limited to the embodiments set forth herein.Given the teaching herein, one of ordinary skill in the art will be ableto modify the invention for a desired purpose and such variations areconsidered within the scope of the invention.

We claim:
 1. A method for the treatment of cancer in a human, whereinthe cancer is Retinoblastoma-positive (Rb) and selected from the groupconsisting of breast cancer, colon cancer, ovarian cancer, non-smallcell lung cancer, prostate cancer, and glioblastoma, comprisingadministering to the human an effective amount of a cyclin dependentkinase 4/6 (CDK4/6) inhibitor of formula:

or a pharmaceutically acceptable salt thereof.
 2. The method of claim 1,wherein the cancer is breast cancer.
 3. The method of claim 1, whereinthe cancer is colon cancer.
 4. The method of claim 1, wherein the canceris ovarian cancer.
 5. The method of claim 1, wherein the cancer isnon-small cell lung cancer.
 6. The method of claim 1, wherein the canceris glioblastoma.
 7. The method of claim 1, wherein the cancer isprostate cancer.
 8. The method of claim 1, wherein the compound isadministered in combination with another chemotherapeutic agent.
 9. Themethod of claim 8, wherein the CDK4/6 inhibitor is administered prior toadministration of the chemotherapeutic agent.
 10. The method of claim 8,wherein the CDK4/6 inhibitor is administered 4 hours or less prior toadministration of the chemotherapeutic agent.
 11. A method for thetreatment of Retinoblastoma-positive breast cancer in a human comprisingadministering an effective amount of a cyclin dependent kinase 4/6(CDK4/6) inhibitor of formula

or a pharmaceutically acceptable salt thereof.
 12. The method of claim11, wherein the compound is administered in combination with achemotherapeutic agent.
 13. The method of claim 12, wherein thechemotherapeutic agent is doxorubicin.
 14. The method of claim 12,wherein the chemotherapeutic agent is paclitaxel.
 15. The method ofclaim 12, wherein the chemotherapeutic agent is carboplatin.
 16. Themethod of claim 12, wherein the chemotherapeutic agent is gemcitabine.17. The method of claim 12, wherein the chemotherapeutic agent iscisplatin.
 18. The method of claim 12, wherein the CDK4/6 inhibitor isadministered prior to administration of the chemotherapeutic agent. 19.The method of claim 12, wherein the CDK4/6 inhibitor is administered 4hours or less prior to administration of the chemotherapeutic agent. 20.The method of claim 11, wherein the breast cancer is estrogen-receptorpositive.
 21. A method for the treatment of Retinoblastoma (Rb)-positivenon-small cell lung cancer in a human comprising administering aneffective amount of a cyclin dependent kinase 4/6 (CDK4/6) inhibitor offormula

or a pharmaceutically acceptable salt thereof.
 22. The method of claim21, wherein the compound is administered in combination with achemotherapeutic agent.
 23. The method of claim 22, wherein thechemotherapeutic agent is docetaxel.
 24. The method of claim 22, whereinthe chemotherapeutic agent is doxorubicin.
 25. The method of claim 22,wherein the chemotherapeutic agent is paclitaxel.
 26. The method ofclaim 22, wherein the chemotherapeutic agent is etoposide.
 27. Themethod of claim 22, wherein the chemotherapeutic agent is vinblastinesulfate.
 28. The method of claim 22, wherein the chemotherapeutic agentis carboplatin.
 29. The method of claim 22, wherein the chemotherapeuticagent is gemcitabine.
 30. The method of claim 22, wherein thechemotherapeutic agent is cisplatin.
 31. A method for the treatment ofcancer in a human, wherein the cancer is Retinoblastoma-positive (Rb)and selected from the group consisting of breast cancer, colon cancer,ovarian cancer, non-small cell lung cancer, prostate cancer, andglioblastoma, comprising: administering to the human an effective amountof a cyclin dependent kinase 4/6 (CDK4/6) inhibitor of formula:

or a pharmaceutically acceptable salt thereof; and administering to thehuman a chemotherapeutic agent; wherein the CDK4/6 inhibitor isadministered to the human prior to administration of thechemotherapeutic agent.
 32. The method of claim 31, wherein the canceris breast cancer.
 33. The method of claim 31, wherein the cancer iscolon cancer.
 34. The method of claim 31, wherein the cancer is ovariancancer.
 35. The method of claim 31, wherein the cancer is non-small celllung cancer.
 36. The method of claim 31, wherein the cancer isglioblastoma.
 37. The method of claim 31, wherein the cancer is prostatecancer.
 38. The method of claim 8, wherein the CDK4/6 inhibitor isadministered 24 hours or less prior to administration of thechemotherapeutic agent.
 39. The method of claim 8, wherein the CDK4/6inhibitor is administered 4 hours or less prior to administration of thechemotherapeutic agent.
 40. A method for the treatment ofRetinoblastoma-positive breast cancer in a human comprising:administering to the human an effective amount of a cyclin dependentkinase 4/6 (CDK4/6) inhibitor of formula:

or a pharmaceutically acceptable salt thereof; and administering to thehuman a chemotherapeutic agent; wherein the CDK4/6 inhibitor isadministered to the human prior to administration of thechemotherapeutic agent.
 41. The method of claim 40, wherein thechemotherapeutic agent is doxorubicin.
 42. The method of claim 40,wherein the chemotherapeutic agent is paclitaxel.
 43. The method ofclaim 40, wherein the chemotherapeutic agent is carboplatin.
 44. Themethod of claim 40, wherein the chemotherapeutic agent is gemcitabine.45. The method of claim 40, wherein the chemotherapeutic agent iscisplatin.
 46. The method of claim 40, wherein the CDK4/6 inhibitor isadministered 24 hours or less prior to administration of thechemotherapeutic agent.
 47. The method of claim 46, wherein the CDK4/6inhibitor is administered 4 hours or less prior to administration of thechemotherapeutic agent.
 48. The method of claim 40, wherein the breastcancer is estrogen-receptor positive.
 49. A method for the treatment ofRetinoblastoma (Rb)-positive non-small cell lung cancer in a humancomprising: administering to the human an effective amount of a cyclindependent kinase 4/6 (CDK4/6) inhibitor of formula:

or a pharmaceutically acceptable salt thereof; and administering to thehuman a chemotherapeutic agent; wherein the CDK4/6 inhibitor isadministered to the human prior to administration of thechemotherapeutic agent.
 50. The method of claim 49, wherein thechemotherapeutic agent is docetaxel.
 51. The method of claim 49, whereinthe chemotherapeutic agent is doxorubicin.
 52. The method of claim 49,wherein the chemotherapeutic agent is paclitaxel.
 53. The method ofclaim 49, wherein the chemotherapeutic agent is etoposide.
 54. Themethod of claim 49, wherein the chemotherapeutic agent is vinblastinesulfate.
 55. The method of claim 49, wherein the chemotherapeutic agentis carboplatin.
 56. The method of claim 49, wherein the chemotherapeuticagent is gemcitabine.
 57. The method of claim 49, wherein thechemotherapeutic agent is cisplatin.
 58. The method of claim 49, whereinthe CDK4/6 inhibitor is administered 24 hours or less prior toadministration of the chemotherapeutic agent.
 59. The method of claim49, wherein the CDK4/6 inhibitor is administered 4 hours or less priorto administration of the chemotherapeutic agent.